Compositions and Methods for Immunomodulation in an Organism

ABSTRACT

The present invention relates to a therapeutic polypeptide and methods for its creation and use for modulating an immune response in a host organism in need thereof. In particular, the invention relates to the administration to an organism in need thereof, of an effective amount of a pre-coupled polypeptide complex comprising a lymphokine polypeptide portion, for example IL-15 (SEQ ID NO: 5, 6), IL-2 (SEQ ID NO: 10, 12) or combinations of both, and an interleukin receptor polypeptide portion, for example IL-15Ra (SEQ ID NO: 7, 8), IL-2Ra (SEQ ID NO: 9, 11) or combinations of both, for augmenting the immune system in, for example, cancer, SCID, AIDS, or vaccination; or inhibiting the immune system in, for example, rheumatoid arthritis, or Lupus. The therapeutic complex of the invention surprisingly demonstrates increased half-life, and efficacy in vivo.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is entitled under 35 U.S.C. § 119(e) to claim thebenefit of U.S. Provisional Patent Application No. 60/681,663, filed May17, 2005, the disclosure of which is hereby incorporated by reference inits entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

The U.S. Government has certain rights in this invention pursuant toGrant No. R01-AI51583 Role of IL-15 in CD8 T Cell Development andResponse, awarded by the National Institutes of Health (NIH).

SEQUENCE LISTING

The present application hereby incorporates by reference, in itsentirety, the Sequence Listing, and identical CRF of the SequenceListing previously filed with the United States Patent and TrademarkOffice in association with U.S. Provisional Patent Application Ser. No.60/681,663; filed: May 17, 2006; entitled: Compositions and Methods forImmunomodulation in an Organism. The CRF contains nucleotide and aminoacid sequences, SEQ. ID NO. 1-16, in file: “IL-15_LLefrancois.txt;”created: May 17, 2005; OS: MS Windows XP; size: 31 KB. In accordancewith 37 CFR 1.821(e) please use the earlier-filed computer readable formfiled in that application as the computer readable form for the instantapplication. An identical paper copy of said Sequence Listing issubmitted, herewith, in the instant application.

FIELD OF THE INVENTION

The present invention relates to a therapeutic polypeptide compositionand methods of administration to an organism in need thereof formodulating immune function. In particular the invention relates to theadministration of an effective amount of a therapeutic protein complexcomprising a lymphokine polypeptide portion, and a lymphokine receptorportion that demonstrates improved in vivo half-life and efficacy whenadministered to an organism.

BACKGROUND

Lymphocytes are a type of white blood cell involved in immune systemregulation. There are two broad categories of lymphocytes, namely Tcells and B cells. T-cells are responsible for cell-mediated immunitywhereas B-cells are responsible for humoral immunity (relating toantibodies). T-cells are named such because these lymphocytes mature inthe thymus and B-cells mature in bone marrow. Lymphocytes are much morecommon in the lymphatic system, and include B cells, T cells, killerT-cells, and natural killer cells. B cells make antibodies that bind topathogens to enable their destruction. CD4+ (helper) T cells co-ordinatethe immune response (they are what become defective in an HIVinfection). CD8+ (cytotoxic) T cells and Natural Killer (NK) cells areable to kill cells of the body that are infected by a virus or displayan antigenic sequence.

Natural killer cells are CD56(+)CD3(−) large granular lymphocytes thatconstitute a key component of the human innate immune response. Inaddition to their potent cytolytic activity, NK cells express a host ofimmunoregulatory cytokines and chemokines that play a crucial role inpathogen clearance. Furthermore, interactions between NK and otherimmune cells are implicated in triggering the adaptive, orantigen-specific, immune response.

The interactions between immune and inflammatory cells are mediated inlarge part by cytokine proteins, for example, lymphokines such asinterleukins (IL), which are able to promote cell growth,differentiation, and functional activation. Currently, at leasttwenty-three interleukins and their various splice variants have beendescribed. Some of these cytokines mediate distinct biological effectsbut many have overlapping activities. The understanding of interleukinstructure and function has led to new and important insights into thefundamental biology of immunity and inflammation. For example,Interleukin-2 (IL-2) and IL-15 are two distinct cytokines with partiallyoverlapping properties that are implicated in the development,homeostasis, and function of T cells and NK cells.

IL-2, formerly referred to as T-cell growth factor, is a powerfulimmunoregulatory lymphokine that is produced by antigen-activated Tcells. It is produced by mature T lymphocytes on stimulation but alsoconstitutively by certain T-cell lymphoma cell lines. IL-2 is useful inthe study of the molecular nature of T-cell differentiation, and becauseit augments natural killer cell activity, it can be useful in modulatingthe immune response to cancers, viral or bacterial infections. Also,IL-2 can act as a growth hormone for both B and T lymphocytes, andstimulates clonal expansion and maturation of these lymphocytes. IL-2binds to its receptor (R) complex comprised of IL-2R alpha (“IL-2Ra”),IL-2R beta (“IL-2Rb”), and -gamma (“gC”) chains, and exerts its effectvia second messengers, mainly tyrosine kinases, which ultimatelystimulate gene expression.

The heterotrimerization of the receptor chains leads to high affinitybinding for IL-2. The functional importance of IL-2Ra in hematopoieticcell systems is well known. However, the potential role that IL-2Raplays in tumorigenesis is still not fully elucidated. IL-2Ra expressionhas been found in many types of cancers, including leukemia, lymphoma,lung, breast, head-and-neck, and prostate. Also, high expression ofIL-2Ra in tumors correlates with a poor prognosis for the patient.

IL-15 is a member of the four alpha-helix bundle family of lymphokinesand its mRNA can be detected in a wide variety of tissues of bothnon-hematopoietic, and hematopoietic lineages but it is not produced byT cells. IL-15 is difficult to detect at the protein level in vivoperhaps due to short protein half-life and tight transcriptional andtranslational control. IL-15 is a soluble protein made by many cells inthe body which play an important role in the development of the immunesystem. IL-15 was simultaneously discovered in an adult T-cell leukemiacell line and a simian kidney epithelial cell line as a 14 kDa-16 kDaprotein able to stimulate cytotoxic T cell lymphocyte cell line (CTLL)and peripheral blood T cell proliferation, and to induce peripheralblood mononuclear cells to exhibit effector function.

IL-15 plays a multifaceted role in development and control of the immunesystem. More specifically, IL-15 influences the function, development,survival, and proliferation of CD8+ T cells, NK cells, killer T cells, Bcells, intestinal intraepithelial lymphocytes (IEL) andantigen-presenting cells (APC). It has been demonstrated that bothIL-15−/−, and IL-15Ra−/− transgenic mice lack peripheral NK and killer Tcell populations, certain IEL subsets, and most memory phenotype CD8+ Tcells. In addition, while antigen-specific memory CD8+ T cells candevelop in response to pathogens in both types of knockout mice, theresulting memory CD8+ T cell pool undergoes dramatic erosion over time.Suggesting a crucial role for IL-15 in mediating long term memory CD8+ Tcell proliferation and survival.

The IL-15 receptor (R) consists of three polypeptides, the type-specificIL-15R alpha (“IL-15Ra”), the IL-2/IL-15Rbeta (“IL-2Rb”), and the commongamma chain (“gC,” which is shared by multiple cytokine receptors). Thehigh affinity IL-15Ra chain (K_(d)≈10⁻¹¹M) is thought to form aheterotrimeric complex with the shared IL-2Rb, and the gC. Similar toIL-15, IL-15Ra is thought to be expressed by a wide variety of celltypes but not necessarily in conjunction with IL-2Rb and gC. Althoughthe IL-15Ra, the IL-2Rb, and the gC chains are believed to associate asa heterotrimeric receptor, whether this is the physiologically relevantform of the IL-15 receptor remains a matter of speculation. For example,the IL-15Ra chain does not co-precipitate with the IL-2Rb/gC in thepresence of IL-15.

Moreover, unlike the IL-2Ra chain, the IL-15Ra chain apparently mediatessignal transduction. IL-15Ra is a 58-60 kDa protein that sharesstructural similarities to the IL-2Ra protein. IL-15Ra and IL-2Ra genesalso share similar intron-exon organization and are closely linked onhuman chromosome 10p14-p15. Human IL-15Ra shares about 45% amino acid(aa) homology with the mouse form of the receptor. Eight isoforms ofIL-15Ra mRNA have been identified resulting from alternative splicingevents involving different exons. The exclusion of exon 2 (ΔExon2)results in an IL-15Ra isoform that does not bind IL-15. HumanIL-15Ra-ΔExon3 cDNA encodes a 267 amino acid (aa) protein that containsa 30 aa signal sequence, a 175 aa extracellular region containing oneN-linked glycosylation site, a 21 aa transmembrane domain and a 41 aacytoplasmic tail.

IL-15 signaling can occur through the heterotrimeric complex of IL-15Ra,IL-2Rb and gC; through the heterodimeric complex of IL-2Rb and gC; orthrough a novel 60-65 kDa IL-15RX subunit found on mast cells.(Anderson, D. M. et al., 1995, J. Biol. Chem. 270:29862-29869;Waldemann, T. A. and Y. Tagaya, 1999, Ann. Rev. Immunol., 17:19-49;Dubois, S. et al., 1999, J. Biol. Chem. 274:26978-26984). Recently, thebinding of IL-15 to IL-15Ra has been reported to antagonize theTNF-alpha-mediated apoptosis in fibroblasts by competing with TNFRI forTRAF2 binding (Bulfone-Paus, S. et al., 1999, FASEB 13:1575-1585).

Given the known effects of IL-15 on the immune system, a number ofgroups have proposed targeting IL-15, to manipulate the immune systemfor the hosts benefit. While IL-15 administration has been employed tobolster immune responses or augment immune system reconstitution,blockade of IL-15 activity can inhibit autoimmune responses. Forexample, administration of an IL-15-activity blocking mutant IL-15-Fcprotein or a soluble form of the IL-15Ra has therapeutic potential in amouse model of arthritis and allograft survival.

Conversely, IL-15 (protein or DNA-expression vector) administered as anadjuvant during vaccination or infection augments CD8+ T cell immunity,and IL-15 treatment can enhance protection of mice from lethal doses ofMycobacterium tuberculosis and Escherichia coli. Furthermore, IL-15therapy stimulates anti-HIV immunity and increases survival of CD4+ andCD8+ lymphocytes from HIV-infected patients in vitro. IL-15 can alsoaccelerate immune reconstitution after bone marrow transplant. Severalgroups have found that IL-15 therapy, in conjunction with chemotherapy,Toll-like receptor agonists, or adoptive transfer of tumor reactive CD8+T cells, can result in increased survival or complete tumor regressionin mouse tumor models, in contrast to each therapy alone. Thus,manipulation of IL-15 activity has potential as a therapeutic modalityin a number of clinical situations.

IL-15 is currently being used in many studies in which augmentation ofthe immune response is desirable. These include increasing the efficacyof vaccines against tumors and infections as well as augmenting theability of the body to remove cancers in the absence of overtvaccination. In addition, IL-15 may aid in regenerating the immunesystem following bone marrow transplant or in AIDS. However, thehalf-life of IL-15 in vivo is very short (minutes to 1 hour or so) andthis is one reason for poor efficacy. At present the only way to obtainany effect of IL-15 activity is by using large doses, and IL-15 alone isnot always effective. Researches have attempted to increase thehalf-life of IL-15 using molecular modifications but these havegenerally been ineffective. For example, PEGylation (a common techniqueto increase protein half-life) of IL-15 increases the half-life butdestroys the majority of the activity of the cytokine, in fact,PEG-IL-15 is an antagonist of IL-15 activity.

Therefore, there exists an unmet need to provide a suitable therapeuticform of IL-15 that demonstrates a longer half-life, and a greaterefficacy at lower dosages when administered to an organism in needthereof for purposes of modulating or enhancing immunity. Such atherapeutic would allow for the administration of less cytokine whilesimultaneously providing for the augmentation of the hosts immune systembeyond the effects of IL-15 alone.

Our studies showed that the IL-15Ra acts to “transpresent” IL-15 toopposing cells expressing the IL-2/15Rb/gC complex without a requirementfor IL-15Ra expression. In addition, in vitro, IL-15 bound to a chimeracomprised of the soluble portion of the IL-15Ra covalently linked to anantibody Fc region (IL-15Ra-Fc) (R&D Systems, Inc., Minneapolis, Minn.),supports the survival of IL-15Ra−/− memory CD8 T cells, in contrast toeither component alone.

It is generally perceived by those in the pertinent art that the solubleportion of the IL-15Ra is an inhibitor of IL-15 action. In fact,published research has demonstrated that IL-15Ra can inhibit IL-15activity in vitro and in vivo. Presently, no one has yet devised asystem in which IL-15 and IL-15Ra are pre-coupled prior toadministration as an in vivo treatment.

SUMMARY OF THE INVENTION

The present invention relates generally to a therapeutic polypeptidecomposition and methods for its administration to an individual in needthereof. The present invention provides nucleic acids and polypeptidesencoded thereby, as well as related compositions including nucleic acidvectors containing the nucleic acids of the invention, cell linescontaining the nucleic acids of the invention, and antibodies (e.g.,polyclonal, monoclonal, chimeric, etc. . . . ) which bind to thetherapeutic polypeptide of the invention. The present invention alsorelates to methods for generating a therapeutic agent comprising atleast one lymphokine or portion thereof, in a pre-coupled complex withat least one lymphokine receptor or portion thereof. It was surprisinglyand unexpectedly observed that the pre-coupled combination of theinvention demonstrates a longer half-life in vivo, and greatertherapeutic efficacy than observed with administration of IL-15 alone.

The invention further encompasses nucleic acid molecules that have atleast 25% homology to the nucleotide sequences shown in SEQ ID NOS: 1-4,and 13-16. It will be appreciated by those skilled in the art that DNAsequence polymorphisms that lead to changes in the amino acid sequencesof the NOVX polypeptides may exist within a population (e.g., the humanpopulation). Such genetic polymorphism in the NOVX genes may exist amongindividuals within a population due to natural allelic variation. Asused herein, the terms “gene” and “recombinant gene” refer to nucleicacid molecules comprising an open reading frame (ORF) encoding aninterleukin and/or interleukin receptor polypeptide, preferably from avertebrate. Such natural allelic variations can typically result in 1-5%variance in the nucleotide sequence. Any and all such nucleotidevariations and resulting amino acid polymorphisms in the polypeptides ofSEQ ID NOs: 5-12, which are the result of natural allelic variation andthat do not alter the functional activity of the polypeptides, areintended to be within the scope of the invention.

In one aspect, the invention relates to nucleic acids and polynucleotidemolecules that encode a lymphokine or portions thereof. In addition, theinvention relates to nucleic acids and polynucleotide molecules thatencode a lymphokine receptor or portions thereof. This aspect of theinvention contemplates the use of polynucleotides that encodesubstantially the full length protein, wild type or mutant polypeptides;discrete segments, domains, subdomains, fragments, deletion or insertionmutations; chimeras; and isoforms and splice variants. This aspect ofthe invention also includes nucleic acids comprising a segment encodingat least one lymphokine or portion thereof, contiguous with a segmentencoding at least one lymphokine receptor or portions thereof within asingle open-reading-frame (ORF). In certain embodiments, the nucleicacids of the invention comprise at least one additional polynucleotidesegment corresponding to transcription regulator sequences (e.g.,promoters, inducible promoters, enhancers, and the like); fusion proteinsequences (e.g., His-tag, GST, GFP, antibody Fc portions, antibioticresistence, signal peptides, and the like); and/or linker sequencesdiposed at the 5′ end, 3′ end or at a location within the polypeptideencoding sequences; and/or combinations thereof. In any of theembodiments described herein, the polynucleotides of the invention mayalso be disposed in a suitable viral vector, bacterial plasmid, orartificial chromosome suitable for cloning and/or expression in aeukaryotic cell or cell extract, prokaryotic cell or cell extract,and/or combinations thereof.

In certain aspects, the present invention relates to a therapeuticcomposition comprising an interleukin polypeptide, for example IL-2 (SEQID NO: 10 and 12), or IL-15 (SEQ ID NO: 5 and 6), including portions andcombinations thereof, in a pre-coupled protein complex with aninterleukin receptor polypeptide, for example IL-2Ra (SEQ ID NO: 9 and11), or IL-15Ra (SEQ ID NO: 7 and 8), including portions andcombinations thereof. In certain embodiments, the invention relates to atherapeutic polypeptide composition comprising a polypeptide having atleast 40% homology to SEQ ID NO.s: 5, 6, 10, 12, portions orcombinations thereof, in a pre-coupled complex with a polypeptide havingat least 40% homology to SEQ ID NO.s: 7, 8, 9, 11, portions orcombinations thereof. In certain other embodiments, the inventionrelates to a therapeutic polypeptide composition comprising apolypeptide having at least 80% homology to SEQ ID NO.s: 5, 6, 10, 12,portions or combinations thereof, in a pre-coupled complex with apolypeptide having at least 80% homology to SEQ ID NO.s: 7, 8, 9, 11,portions or combinations thereof.

In another aspect, the invention relates to the use of a chimericpolypeptides in the polypeptide complex of the invention. In certainembodiments, the invention comprises chimeric polypeptides comprisingone or more interleukins, interleukin receptor, portions andcombinations thereof. In other embodiments, the invention compriseschimeric polypeptides comprising at least one interleukin receptorpolypeptide or portion thereof, for example, the soluble portion of aninterleukin receptor and/or the ligand binding domain, covalently linkedand contiguous with the Fc portion of an antibody. The chimericmolecules of the invention may be synthesized recombinantly byexpressing a polynucleotide containing the desired elements within asingle ORF in any number of combinations, which will be recognized byone of ordinary skill in the art. Other chimeric polypeptides, forexample, a human IL-15Ra (1Met-94Ile)-K-(129Pro-205Thr)-linker-Fcpolypeptide, are commercially available from R&D Systems (Minneapolis,Minn.).

In another aspect the chimeric polynucleotide molecules are contained ina nucleic acid vector, such as for example a plasmid or viral DNAconstruct, for subcloning, expression, purification or other routinegenetic manipulation suitable for use in a eukaryotic or prokaryoticcell or organism. In addition, the chimeric polynucleotide molecules mayoptionally contain additional coding or non-coding sequences, insertedby genetic manipulation in between regions coding for lymphokine orlymphokine receptor portions. In one embodiment, the nucleic acidencoding the interleukin or portion thereof, is disposed in tandemlinkage with a interleukin receptor portion. In still furtherembodiments, a linker sequence is inserted between the terminal codon ofthe first nucleic acid and the first codon of the second nucleic acid.These linkers may be of any length and type suitable, and may be used,for example, to reduce steric constraints on polypeptide folding,introduce a protease or nuclease cleavage site, provide a convenientsite for chemical modification, conjugation or other functional element.

In preferred embodiments, the candidate protein is a human protein. Inother embodiments, the candidate protein is a eukaryotic protein, forexample, a mammalian protein, or a mouse protein. In another aspect, theinvention features a transgenic cell or organism that contains atransgene encoding a an interleukin, an interleukin receptor or portionthereof, and/or a chimeric interleukin/interleukin receptor polypeptide.In another aspect, the invention relates to one or more geneticallyaltered cell lines that contain the polynucleotide constructs of theinvention, such as, for example, by incorporation into the genomic DNAof the cell, retention episomally or as part of an artificialchromosome. In a related aspect, the present invention relates to theexpression by a modified host cell of a nucleic acid encoding individualcomponents or the entire polypeptide complex of the invention. In someembodiments, the transgene encodes a protein that is normally exogenousto the transgenic cell. In some embodiments, the transgene encodes ahuman protein. In some embodiments, the transgene is linked to aheterologous promoter. In other embodiments, the transgene is linked toits native promoter.

In another aspect, the invention relates to antibodies, for example,polyclonal, monoclonal, or chimeric, that recognize and bind to discreteepitopes of the polypeptide complex of the invention or componentsthereof. In certain aspects, the invention relates to the administrationof antibodies specific to the components of the complex of theinvention, the complex of the invention, to other lymphokines, otherlymphokine receptors or combinations thereof. In one embodiment, theinvention comprises an interleukin, for example IL-2, IL-7, or IL-15,pre-coupled to a antibody specific for said interleukin. In otherembodiments, the methods of the invention include method for treating adisease in an individual comprising administering an effective amount ofa pre-coupled complex comprising an interleukin and an antibody specificfor said interleukin to an individual in need thereof.

In another aspect, the present invention relates to methods forproducing an immunomodulatory therapeutic comprising a pre-coupledcomplex of at least one lymphokine polypeptide or portion thereof, andat least one lymphokine receptor polypeptide or portion thereof. Incertain embodiments, the invention includes methods for creating thecomplex of the invention in vitro comprising expressing or synthesizingof component polypeptides, isolating the polypeptides, purifiying and/orconcentrating the polypeptides, and forming of the complex. In thisaspect, the invention relates to creating the pre-coupled polypeptidecomplex of the invention from polypeptides isolated from a host cell orcell extract in which each polypeptide component of the complex isexpressed from two discrete nucleid acids or as a single open readingfrom comprising a chimera comprising the interleukin and interleukinreceptor linked, in frame, in tandem. The purification can be performedby chromatographic means known to one of ordinary skill in the art andmay include, for example, affinity purification, size exclusion, ionexchange, hydroxyapatite, HPLC, and the like.

In another aspect, the invention relates to methods for inducing,enhancing or inhibiting immune cell activity and proliferationcomprising administering an effective amount of a pre-coupledpolypeptide complex to an individual in need thereof, wherein thepre-coupled polypeptide complex comprises at least one lymphokine orportion thereof and at least one lymphokine receptor or portion thereof.In a related aspect of the invention, the complex may be used to augmenta host organism's immunity or immune response to antigen, such as forexample, a bacteria, a virus, a protein, a peptide, a nucleic acid, andthe like. All of the preceding objects of the invention contemplate theuse of IL-15, IL-2, IL-15Ra, or IL-2Ra polypeptides, portions, andcombinations thereof. In yet another aspect, the invention includesmethods of treating an organism, comprising administering to an organisma vector encoding one or more of SEQ ID NO: 1-4, and 13-16.

In another aspect, the invention relates to a pm-coupled polypeptidecomplex useful for increasing the proliferation and survival of memory Tcells, B cells, and NK cells. As such, administration of the therapeuticof the present invention can also be used to enhance pre-existingimmunity (e.g. previously vaccinated individuals) without the need foran actual vaccine booster. In certain aspects the therapeutic of theinvention is administered, for example, for the augmentation ofvaccination, for enhancing immunity in SCID or AIDS patients, and forthe treatment of cancers.

In still other aspects, the pre-coupled polypeptide complex of theinvention is useful for inhibiting a host organism's immune response incases where it is a detriment to the organism. For example, the complexof the invention may be used to inhibit a host organism's immunity orimmune response to antigen, for example an auto-antigen, as observed inindividuals suffering from autoimmune diseases and conditions likerheumatoid arthritis or Lupus. In certain embodiments of this aspect ofthe invention the pre-coupled polypeptide complex comprises lymphokineand lymphokine receptor polypeptides or portions thereof, which areunable to activate immune cells or stimulate their proliferation. Forexample, the polypeptide components of the complex may containmutations, deletions, insertions or chemical modifications that inhibitsignaling via the IL-2 or IL-15 pathways.

In any of the above-described aspects, the pre-coupled polypeptidecomplex can be administered in any pharmaceutically acceptable form(e.g., liquid, powder, pill, controlled release formula, etc. . . . ),via any suitable route (e.g., intravenous, oral, parenteral, subdermal,topical, anal, nasal, etc. . . . ), and optionally with anypharmaceutically acceptable excipients, carriers, and/or in combinationwith other active ingredients (e.g., NSAIDS, Immunosuppressants,Anti-histamines, Anti-oncogenics, Antibiotics, Sulfonamides, etc. . . .). The preceeding are given by way of non-limiting example, and theparticular formulation may vary in any number of ways, which areexpressly incorporated herein, depending on a multitude of factors whichwill be recognizable by one of ordinary skill in the art.

In yet another aspect the present invention relates to a kit comprisinga suitable container, the pre-coupled polypeptide complex of theinvention or the components therefore in a pharmaceutically acceptableform disposed therein, and instructions for its use.

In another aspect, the current invention relates to the production oflibraries containing mutated and modified nucleic acids for use in themethods described, and the nucleic acids identified therein.

In another aspect, the invention relates to a method of detecting thepresence of a lymphokine-lymphokine receptor polypeptide complex in asample. In the method, a sample is contacted with a compound or antibodythat selectively binds under conditions allowing for formation of acomplex between the polypeptide. The complex is detected, if present,thereby identifying the polypeptide complex within the sample. Alsoincluded in the invention is a method of detecting the presence of alymphokine-lymphokine receptor chimeric nucleic acid molecule in asample by contacting the sample with a lymphokine or lymphokine receptornucleic acid probe or primer, and detecting whether the nucleic acidprobe or primer bound to a lymphokine-lymphokine receptor chimericnucleic acid molecule the sample.

Additional advantageous features and functionalities associated with thesystems, methods and processes of the present invention will be apparentfrom the detailed description which follows. The publications and othermaterials used herein to illuminate the background of the invention, andin particular cases, to provide additional details respecting thepractice, are incorporated by reference, and for convenience are listedin the appended bibliography.

DESCRIPTION OF THE DRAWINGS

FIG. 1. Co-administration of pre-coupled IL-15+ IL-15Ra-Fc enhances CD8+T cell proliferative response to exogenous IL-15. On day-1 mice receivedabout 1×10⁷ congenic CFSE-labeled, CD8-enriched lymphocytes i.v. andwere treated i.p. on day 0 with PBS; IL-15 (about 2.5 μg); or IL-15Ra-Fc(about 15 μg) with IL-15 (2.5 μg). CD8+ splenocytes were analyzed on day4 by flow cytometry for CFSE fluorescence and CD45.1 expression (toppanels); or CD45.1+ CD8+ cells were analyzed for CFSE fluorescence andCD44 expression (middle panels). Bottom panels: On day-1 mice receivedCFSE-labeled CD8+ T cell enriched splenocytes containing about 6.5×10⁵tetramer+ OVA-specific memory CD8+ T cells and were treated on day 0with PBS, IL-15 (about 2.5 μg) or IL-15Ra-Fc (about 15 μg) with IL-15(about 2.5 μg). Donor tetramer+ splenocytes were analyzed by flowcytometry on day 4 for CFSE fluorescence. IL-15Ra-Fc treatment alone hadno effect on proliferation (data not shown). Data are representative of3 similar experiments with 3 mice per group.

FIG. 2. NK cells are highly responsive to pre-coupled IL-15+IL-15Ra-Fc.On day-1, mice received about 1.5×10⁷ congenic CFSE-labeled lymphocytesi.v. and on day 0 were treated with PBS, IL-15 (about 2.5 μg), or IL-15(about 2.5 μg)+IL-15Ra-Fc (about 15 μg) i.p. Spleen cells were analyzedby flow cytometry on day 4. Samples were gated on the indicated in thedonor population. Data is representative of 2 experiments with 3 miceper group.

FIG. 3. CD8+ T cells rapidly divide in response to pre-coupledIL-15+IL-15Ra-Fc treatment. On day-1 mice received about 1×10⁷ congenicCFSE-labeled, CD8 enriched lymphocytes i.v. and were treated with PBS orIL-15 (about 2.5 μg)+IL-15Ra-Fc (about 15 μg) on day 0. Peripheral bloodlymphocytes were analyzed by flow cytometry on days 1-4. Samples shownare gated on live donor CD8 T cells. Data are representative of 2experiments with at least 3 mice per group. PBS treatment had no effecton cell division (data not shown).

FIG. 4. Coadministration of IL-15Ra-Fc with IL-15 greatly enhances IL-15potency. (a) On day-1 mice received about 1.5×10⁶ congenic CFSE-labeled,CD8 enriched lymphocytes i.v. and on day 0 received either PBS (notshown), IL-15 (about 5 μg) or varying doses of IL-15 with IL-15Ra-Fc(about 2.5 μg+15 μg, about 0.5 μg+3 μg, about 0.1 μg+0.6 μg, or about0.02 μg+0.12 μg) i.p. (b) On day-1 each mouse received about 4.5×10⁶congenic CFSE-labeled, CD8 enriched lymphocytes i.v. and on day 0received either PBS (not shown), IL-15 (about 0.5 μg)+IL-15Ra-Fc (about3 μg), or varying doses of IL-15 (about 12.5 μg, 25 μg, or 37.5 μg) i.p.CD8+ splenocytes were analyzed on day 4 for CFSE dilution by flowcytometry.

FIG. 5. Activity of complexed IL-15+IL-15Ra-Fc requires IL-2Ra but notIL-15Ra expression by responding cells. (a) On day-1 IL-15Ra−/− micereceived congenic CFSE-labeled, CD8 enriched IL-15Ra−/− lymphocytes i.v.and on day 0 were treated with PBS, IL-15 (about 2.5 μg) or IL-15 (about2.5 μg)+IL-15Ra-Fc (about 15 μg) i.p. On day 4 CD8+ donor splenocyteswere analyzed for CFSE fluorescence and CD44 and CD122 expression. (b)On day-1 normal mice received congenic CFSE-labeled wild type orIL-2/IL-15Ra−/− splenocytes i.v. and on day 0 were treated with eitherPBS or IL-15 (about 2.5 μg)+IL-15Ra-Fc (about 15 μg) i.p. CD8+ donorsplenocytes were analyzed for CFSE dilution on day 4 by flow cytometry.

FIG. 6. Pre-coupled IL-15+IL-15Ra-Fc driven proliferation of CD8+ Tcells requires MHC class I expression, but does not require IL-7 or DC.(a) On day-1 B6 and beta2m−/− mice received a mixture of normal B6 andnaïve OT-I-RAG−/− CFSE-labeled CD8+ T cells and on day 0 were treatedwith either PBS or IL-15 (about 2.5 μg)+IL-15Ra-Fc (about 15 μg). (b) Onday-1 IL-7+/− or IL-7−/− mice received congenic CFSE-labeledCDS-enriched lymphocytes. On day 0, mice received IL-15 (about 2.5μg)+IL-15Ra-Fc (about 15 μg) i.p. (c) On day-1 chimeras produced with B6or CD11c-DTR bone marrow received splenocytes i.v and on day 0 weretreated with either PBS or IL-15Ra-Fc (about 15 μg)+IL-15 (about 2.5 μg)i.p. All mice were treated with DT on days 0,1, and 3. In all casesdonor CD8+ splenocytes were analyzed for CFSE dilution on day 4.

FIG. 7. Naïve CD8+ T cells acquire effector phenotype and function inresponse to pre-coupled IL-15+IL-15Ra-Fc treatment. On day-1 micereceived a mixture of naïve and memory OT-I-RAG−/− cells (a) or onlynaïve OT-I-RAG−/− cells (b-d), and were treated with either PBS orrmIL-15Ra-Fc (about 15 μg) with IL-15 (about 2.5 μg) on day 0. Four dayslater splenocytes were examined for (a) CFSE intensity, (b) percentageof donor OT-I and CD44 expression. (c) On day-1 mice received about7×10⁵ naïve OT-I-RAG−/− cells and on day 0 were treated with PBS, IL-15(about 2.5 μg), IL-15 (about 2.5 μg)+IL-15Ra-Fc (about 15 μg), or about1×10⁵ pfu VSV-OVA. On day 4 splenocytes were incubated in vitro with orwithout SIINFEKL peptide for about 5 hours and the production of IFN-a̧was analyzed by intracellular staining. (d) On day-1 mice received about2×10⁶ naïve OT-I-RAG−/− cells and were treated with PBS or IL-15 (about2.5 μg)+IL-15Ra-Fc (about 15 μg) i.p or about 1×10⁵ pfu of VSV-OVA i.v.On day 4 posttreatment each mouse received a mixture of CFSE-labeled(about 0.25 μM) non-peptide pulsed splenocytes and CFSE-labeled (about2.5 μM) SIINFEKL peptide pulsed splenocytes. Four hours latersplenocytes were analyzed for the presence of the CFSE-labeled targetpopulations.

FIG. 8. Pre-coupled IL-15+IL-15Ra-Fc treatment generates memory cellsfrom naïve CD8+ T cells. On day one, B6 mice received about 6×10⁶CFSE-labeled naïve OT-I-RAG−/− cells and on day 0 were treated i.p. withPBS or IL-15 (about 2.5 μg)+IL-15Ra-Fc (about 15 μg). 44 days latersplenocytes were analyzed for percentage of donor OT-I CD8+ T cells (toppanels) and OT-I expression of CD44 and CD122 (middle and bottompanels).

FIG. 9. Example of a IL-15+IL-15Ra-Fc fusion protein of theInvention—mouse version of the fusion protein. In this example, thegeneral construct includes an IL-2 signal peptide for enhancedexpression and processing, the IL-15 gene or portion thereof, a variablelinker region to promote steric freedom and protein folding (may be ofany desired length or sequence), the soluble or extracellular portion ofthe IL-15Ra gene, and the Fc portion of a human IgG. The genes orportions thereof of the human homologs can be substituted in a similarfashion. Similarly, IL-2 and IL-2Ra genes or portions thereof can besubstituted in a chimeric construct, which also includes combinationswith IL-15 or IL-15Ra genes or portions thereof.

FIG. 10. The IL-15+IL-15Ra fusion protein elicits proliferation of CD8+T cells and NK Cells. CFSE-labeled lymphocytes were transferred tonormal mice that were then treated with ˜10 μg of the Il-15+IL-15Rafusion protein. Four days later, spleen cells were isolated and analyzedby flow cytometry.

FIG. 11. Liver Cancer Burden Reduced by IL-15+IL-15Ra Protein Complex inMice. About 1×10⁵ B6-F1 melanoma cells were injected intrasplenically(which directs tumors to the liver). On days 1 and 7 days later micewere treated with PBS (control), 2.5 μg IL-15 or 2.5 μg IL-15+IL-15Racomplex. Fourteen days after inoculation tumors were counted in theliver and the spleens were weighed.

FIG. 12. Complexing IL-15 to IL-15Ra greatly enhances half-life andbioavailability in vivo. (A) 2.5 μg of human IL-15 alone orpre-complexed to IL-15Ra was administered to mice by intraperitonealinjection. At the indicated times, serum was obtained and tested byELISA for the presence of IL-15. The total IL-15 present was calculatedfrom a standard concentration curve. (B) Half-life was calculated fromthe linear portion of the decay curve in A.

DETAILED DESCRIPTION OF THE INVENTION

In examples of the compositions and methods of preferred embodiments,the useful and advantageous generation of therapeutic polypeptides ispresented. In a preferred embodiment, the invention relates to atherapeutic polypeptide complex comprising a lymphokine or portionsthereof, and a lymphokine receptor or portions thereof. The term“lymphokine receptor” or “interleukin receptor” refers to thetransmembrane receptors for a respective lymphokine or interleukin, andin some embodiments may comprise an antibody capable of binding saidlymphokine or interkeukin. In this context the antibody functionseffectively as the “receptor” for the lymphokine or interleukinpolypeptide.

Without being restricted to any particular theory, the inventorshypothesize that the activity of the therapeutic of the inventionresults from a process termed, “trans-presentation” in which thereceptor portion of the polypeptide complex functions to present thesignaling molecule portion to its respective receptor(s) on the targetcell's surface. For example, experimental evidence indicates thatIL-15Ra trans-presents IL-15 to T cells and other cells in vivo throughthe beta and gamma chains of the IL-15 receptor. The theory is supportedby in vivo results in mice that show IL-15 alone had little activity butthe pre-coupled IL-15+IL-15Ra complex had substantial activity ondriving memory T cell proliferation (one of the hallmarks of IL-15activity) as shown in FIG. 1, and reducing tumor burden Table 1. Bypre-coupling IL-15 to the IL-15Ra chain the biological activity of IL-15was greatly augmented in mice (FIGS. 1-8, and 10-12).

Unless clearly indicated to the contrary, the following definitionssupplement definitions of terms known in the art.

The term “nucleic acid” refers to deoxyribonucleotides, deoxyribonucleicacids, ribonucleotides, and ribonucleic acids, and polymeric formsthereof, and includes either single- or double-stranded forms. Also,unless expressly limited, the term “nucleic acid” includes knownanalogues of natural nucleotides, for example, peptide nucleic acids(“PNA”s), that have similar binding properties as the reference nucleicacid. In addition, in any of the preferred embodiments, a particularnucleotide or nucleic acid sequence includes conservative variations(e.g. degenerate codon substitutions; see below), complementarysequences, as well as the sequence explicitly indicated. A degeneratecodon substitution is one in which the third position of one or moreselected codons is substituted with any nucleotide which results in thesame amino acid. The term nucleic acid is generic to the terms “gene,”“DNA,” “cDNA,” “oligonucleotide,” “RNA,” “mRNA,” “nucleotide,”“polynucleotide,” and the like.

As used herein, the term “oligonucleotide” refers to a series of linkednucleotide residues. A short oligonucleotide sequence may be based on,or designed from, a genomic or cDNA sequence and is used to amplify,confirm, or reveal the presence of an identical, similar orcomplementary DNA or RNA in a particular cell or tissue.Oligonucleotides comprise a nucleic acid sequence having about 10 nt, 50nt, or 100 nt in length, preferably about 15 nt to 30 nt in length. Inone embodiment of the invention, an oligonucleotide comprising a nucleicacid molecule less than 100 nt in length would further comprise at least6 contiguous nucleotides of SEQ ID NOS: 1-4, or 13-16. Oligonucleotidesmay be chemically synthesized and may also be used as probes.

A “recombinant” nucleic acid is any nucleic acid produced by an in vitroor artificial (meaning not naturally occurring) process or byrecombination of two or more nucleic acids.

The term “gene” is used broadly to refer to any segment of nucleic acidassociated with expression of a given RNA or protein. Thus, genesinclude regions encoding expressed RNAs (which typically includepolypeptide coding sequences) and, often, the regulatory sequencesrequired for their expression. Genes can be obtained from a variety ofsources, including cloning from a source of interest or synthesizingfrom known or predicted sequence information, and may include sequencesdesigned to have specifically desired parameters.

In another embodiment, an isolated nucleic acid molecule of theinvention comprises a nucleic acid molecule that is a complement of thenucleotide sequence shown in SEQ ID NOs: 1-4, and 13-16. As used herein,the term “complementary” refers to Watson-Crick or Hoogsteen basepairing between nucleotides units of a nucleic acid molecule, and theterm “binding” means the physical or chemical interaction between twopolypeptides or compounds or associated polypeptides or compounds orcombinations thereof. Binding includes ionic, non-ionic, van der Waals,hydrophobic interactions, and the like. A physical interaction can beeither direct or indirect. Indirect interactions may be through or dueto the effects of another polypeptide or compound. Direct binding refersto interactions that do not take place through, or due to, the effect ofanother polypeptide or compound, but instead are without othersubstantial chemical intermediates. “Fragments” provided herein aredefined as sequences of at least 6 (contiguous) nucleic acids or atleast 4 (contiguous) amino acids, a length sufficient to allow forspecific hybridization in the case of nucleic acids or for specificrecognition of an epitope in the case of amino acids, and are at mostsome portion less than a full length sequence. Fragments may be derivedfrom any contiguous portion of a nucleic acid or amino acid sequence ofchoice. A full-length clone is identified as containing an ATGtranslation start codon and an in-frame stop codon. Any disclosed NOVXnucleotide sequence lacking an ATG start codon therefore encodes atruncated C-terminal fragment of the respective polypeptide, andrequires that the corresponding full-length cDNA extend in the 5′direction of the disclosed sequence. Any disclosed nucleotide sequencelacking an in-frame stop codon similarly encodes a truncated N-terminalfragment of the respective polypeptide, and requires that thecorresponding full-length cDNA extend in the 3′ direction of thedisclosed sequence.

The term “host cell” includes a cell that might be used to carry aheterologous nucleic acid, or expresses a peptide or protein encoded bya heterologous nucleic acid. A host cell can contain genes that are notfound within the native (non-recombinant) form of the cell, genes foundin the native form of the cell where the genes are modified andre-introduced into the cell by artificial means, or a nucleic acidendogenous to the cell that has been artificially modified withoutremoving the nucleic acid from the cell. A host cell may be eukaryoticor prokaryotic. For example, bacteria cells may be used to carry orclone nucleic acid sequences or express polypeptides. General growthconditions necessary for the culture of bacteria can be found in textssuch as BERGEY'S MANUAL OF SYSTEMATIC BACTERIOLOGY, Vol. 1, N. R. Krieg,ed., Williams and Wilkins, Baltimore/London (1984). A “host cell” canalso be one in which the endogenous genes or promoters or both have beenmodified to produce one or more of the polypeptide components of thecomplex of the invention.

“Derivatives” are nucleic acid sequences or amino acid sequences formedfrom the native compounds either directly, by modification, or bypartial substitution. “Analogs” are nucleic acid sequences or amino acidsequences that have a structure similar to, but not identical to, thenative compound, e.g. they differ from it in respect to certaincomponents or side chains. Analogs may be synthetic or derived from adifferent evolutionary origin and may have a similar or oppositemetabolic activity compared to wild type. Homologs are nucleic acidsequences or amino acid sequences of a particular gene that are derivedfrom different species.

Derivatives and analogs may be full length or other than full length.Derivatives or analogs of the nucleic acids or proteins of the inventioninclude, but are not limited to, molecules comprising regions that aresubstantially homologous to the nucleic acids or proteins of theinvention, in various embodiments, by at least about 30%, 45%, 70%, 80%,or 95% identity (with a preferred identity of 80-95%) over a nucleicacid or amino acid sequence of identical size or when compared to analigned sequence in which the alignment is done by a computer homologyprogram known in the art, or whose encoding nucleic acid is capable ofhybridizing to the complement of a sequence encoding the proteins of theinvention under stringent, moderately stringent, or low stringentconditions. See e.g. Ausubel, et al., CURRENT PROTOCOLS IN MOLECULARBIOLOGY, John Wiley & Sons, New York, N.Y., 1993. Nucleic acidderivatives and modifications include those obtained by genereplacement, site-specific mutation, deletion, insertion, recombination,repair, shuffling, endonuclease digestion, PCR, subcloning, and relatedtechniques.

“Homologs” can be naturally occurring, or created by artificialsynthesis of one or more nucleic acids having related sequences, or bymodification of one or more nucleic acid to produce related nucleicacids. Nucleic acids are homologous when they are derived, naturally orartificially, from a common ancestor sequence (e.g., orthologs orparalogs). If the homology between two nucleic acids is not expresslydescribed, homology can be inferred by a nucleic acid comparison betweentwo or more sequences. If the sequences demonstrate some degree ofsequence similarity, for example, greater than about 30% at the primaryamino acid structure level, it is concluded that they share a commonancestor. The degree of similarity will vary and important factorsinclude for example, the degree of overall similarity, the degree ofsimilarity within specific regions of the coding sequence, thesimilarity of noncoding sequence, and the activity of the polypeptide.For purposes of the present invention, genes are homologous if thenucleic acid sequences are sufficiently similar to allow recombination.

The terms “homology” or “identity,” in the context of two or morenucleic acid or polypeptide sequences, refer to two or more sequences orsubsequences that are the same or similar, and have a specifiedpercentage of amino acid residues or nucleotides that are the same, whencompared and aligned for maximum correspondence, as measured using oneof the sequence comparison algorithms such as BLAST, ClustalW, or otheralgorithms available to persons of skill or by visual inspection. Forsequence comparison and homology determination, typically one sequenceacts as a reference sequence to which test sequences are compared. Whenusing a sequence comparison algorithm, test and reference sequences areinput into a computer, subsequence coordinates are designated, ifnecessary, and sequence algorithm program parameters are designated. Thesequence comparison algorithm then calculates the percent sequenceidentity for the test sequence(s) relative to the reference sequence,based on the designated program parameters. Other determinations ofhomology include hybridization of nucleic acids under stringentconditions.

The phrase “hybridizing,” refers to the binding, duplexing, orhybridizing of a molecule only to a particular nucleotide sequence understringent conditions, including when that sequence is present in acomplex mixture (e.g., total cellular) DNA or RNA.

The term “pre-coupled” as used herein, refers to a situation whereindividual polypeptide components are combined to form the activecomplex prior to activation or binding at the target site, for example,an immune cell. This includes the situation where the individualpolypeptide complex components are synthesized or recombinantlyexpressed and subsequently isolated and combined to form a complex, invitro, prior to administration to an organism; the situation where achimeric or fusion polypeptide (i.e., each discrete protein component ofthe complex is contained in a single polypeptide chain) is synthesizedor recombinantly expressed as an intact complex; and/or the situationwhere individual polypeptide complex components are administeredsimultaneously to an individual, for example, intravenously, and formcomplexes in situ or in vivo.

“Conservative mutations” of a nucleic acid sequence refers to thosenucleotides that encode identical or essentially identical amino acidsequences, or where the nucleotide does not encode an amino acidsequence, to essentially identical sequences. This is based on the factthat the genetic code is “degenerate,” that is to say a number ofdistinct nucleic acids encode for the same amino acid. For instance, thecodons GTT, GTA, GTC, and GTG all encode the amino acid valine. Thus, atevery position where a valine is specified by a codon, the codon can bealtered to any of the corresponding codons described without alteringthe encoded polypeptide. Such nucleic acid variations are “silentmutations,” which are one species of “conservative mutation.” Unlessotherwise described every nucleotide sequence described herein whichencodes an amino acid also includes every possible silent variation. Oneof ordinary skill will recognize that each codon in a nucleic acid(except ATG, which is ordinarily the only codon for methionine) can bemodified to yield a functionally identical molecule by standardtechniques. Accordingly, in each instance where mutagenesis is used each“silent mutation” of a nucleic acid, which encodes an amino acid, isimplicitly included.

Furthermore, one of ordinary skill will recognize that “conservativemutations” also include the substitution, deletion or addition ofnucleic acids that alter, add or delete a single amino acid or a smallnumber of amino acids in a coding sequence where the nucleic acidalterations result in the substitution of a chemically similar aminoacid. Amino acids that may serve as conservative substitutions for eachother include the following: Basic: Arginine (R), Lysine (K), Histidine(H); Acidic: Aspartic acid (D), Glutamic acid (E), Asparagine (N),Glutamine (Q); hydrophilic: Glycine (G), Alanine (A), Valine (V),Leucine (L), Isoleucine (I); Hydrophobic: Phenylalanine (F), Tyrosine(Y), Tryptophan (W); Sulfur-containing: Methionine (M), Cysteine (C). Inaddition, sequences that differ by conservative variations are generallyhomologous.

A “subsequence” refers to a sequence of nucleic acids or amino acidsthat comprise a part of a longer sequence of nucleic acids or aminoacids (e.g., polypeptide) respectively.

A nucleic acid “operon” includes a gene that is situated in a functionalrelationship with other nucleic acid sequences, for example, a promoter,an enhancer, termination signals, or another gene if it increases thetranscription of the coding sequence.

“Mutagenesis” as used herein includes such techniques known in the artas PCR mutagenesis, oligonucleotide-directed mutagenesis, site-directedmutagenesis, random mutagenesis, error-prone PCR mutagenesis, etc., andreiterative sequence recombination by any of the techniques describedherein.

Descriptions of the molecular biological techniques useful to thepractice of the invention including mutagenesis, PCR, cloning, and thelike include Berger and Kimmel, GUIDE TO MOLECULAR CLONING TECHNIQUES,METHODS IN ENZYMOLOGY, volume 152, Academic Press, Inc., San Diego,Calif. (Berger); Sambrook et al., MOLECULAR CLONING—A LABORATORY MANUAL(2nd Ed.), Vol. 1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor,N.Y., 1989, and CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, F. M. Ausubel etal., eds., Current Protocols, a joint venture between Greene PublishingAssociates, Inc. and John Wiley & Sons, Inc.; Berger, Sambrook, andAusubel, as well as Mullis et al., U.S. Pat. No. 4,683,202 (1987); PCRPROTOCOLS A GUIDE TO METHODS AND APPLICATIONS (Innis et al. eds),Academic Press, Inc., San Diego, Calif. (1990) (Innis); Arnheim &Levinson (Oct. 1, 1990) C&EN 36-47; Lueng, et al., A method for randommutagenesis of a defined DNA segment using a modified polymerase chainreaction. Technique: J Methods Cell Molec Biol 1(1):11-15 (1989), whichare incorporated herein by reference in their entirety for all purposes.Exemplary methods of the present invention include performing sequencemutagenesis, recombination, or both, and screening or selection ofindividual genes.

As used herein, the terms “lymphokine,” “interleukin,” “IL-15,” or“IL-2” is used to refer collectively to all forms of the correspondingpolynucleotide or polypeptide sequences including, for example the fulllength sequence, segments, domains or discrete portions, substitutions,insertion and deletion mutants, chimeras with the same or otherlymphokines, isoforms, splice variants, and any combinations thereof.

As used herein, the terms “IL-15Ra,” or “IL-2Ra” is used to refercollectively to all forms of the corresponding polynucleotide orpolypeptide sequences including, for example the full length sequence,segments, domains or discrete portions, substitutions, insertion anddeletion mutants, chimeras with the same or other lymphokine receptors,isoforms, splice variants, and any combinations thereof.

Nucleic Acid Molecules

In some embodiments, the invention comprises nucleic acids andpolynucleotide molecules that encode a lymphokine or portions thereof,and nucleic acids and polynucleotide molecules that encode a lymphokinereceptor or portions thereof. In any of the nucleic acid embodiments,the invention contemplates the use of polynucleotides that encodesubstantially the full length protein, wild type or mutant polypeptides;discrete segments, domains, subdomains, fragments, deletion or insertionmutations; chimeras; and isoforms and splice variants. In certain of thepreferred embodiments, the invention comprises nucleic acids comprisinga polynucleotide segment encoding at least one lymphokine or portionthereof, contiguous with a polynucleotide segment encoding at least onelymphokine receptor or portion thereof within a singleopen-reading-frame or ORF (i.e., start codon to stop codon). In certainembodiments, the nucleic acids of the invention comprise at least oneadditional polynucleotide segment comprising a transcription regulatorysequences (e.g., promoters, inducible promoters, enhancers, and thelike); fusion protein sequences (e.g., His-tag, GST, GFP, antibody Fcportions, antibiotic resistence, signal peptides, and the like); and/orlinker sequences diposed at the 5′ end, 3′ end or at a location withinthe polypeptide encoding sequences; and/or combinations thereof. In anyof the embodiments described herein, the polynucleotides of theinvention may also be disposed in a suitable viral vector, bacterialplasmid, or artificial chromosome suitable for cloning and/or expressionin a eukaryotic cell or cell extract, prokaryotic cell or cell extract,and/or combinations thereof.

Many techniques for the cloning, subcloning, and transfer of recombinantnucleic acids into a plasmid vector or a host cell or both, andtechniques for library screening and selection, are known in the art,and each of these formats and/or techniques is generally applicable tothe present invention. For example, texts that disclose generaltechniques for manipulating nucleic acids of use in this inventioninclude “Current Protocols in Molecular Biology” (Ausubel et al., eds.,1994)); Sambrook et al., “Molecular Cloning, A Laboratory Manual” (2nded. 1989); and Kriegler, “Gene Transfer and Expression: A LaboratoryManual” (1990), the contents and relevant teachings of which are herebyincorporated by reference.

Another aspect of the invention pertains to vectors, preferablyexpression vectors, containing a nucleic acid encoding SEQ ID NOs: 5-12,or derivatives, fragments or homologs thereof. As used herein, the term“vector” refers to a nucleic acid molecule capable of transportinganother nucleic acid to which it has been “operably linked.” One type ofvector is a “plasmid”, which refers to a circular double stranded DNAloop into which additional DNA segments can be ligated. Another type ofvector is a viral vector, wherein additional DNA segments can be ligatedinto the viral genome. Certain vectors are capable of autonomousreplication in a host cell into which they are introduced (e.g.,bacterial vectors having a bacterial origin of replication and episomalmammalian vectors). Other vectors (e.g., non-episomal mammalian vectors)are integrated into the genome of a host cell upon introduction into thehost cell, and thereby are replicated along with the host genome.Moreover, certain vectors are capable of directing the expression ofgenes to which they are operatively-linked. Such vectors are referred toherein as “expression vectors”. In general, expression vectors ofutility in recombinant DNA techniques are often in the form of plasmids.In the present specification, “plasmid” and “vector” can be usedinterchangeably as the plasmid is the most commonly used form of vector.However, the invention is intended to include such other forms ofexpression vectors, such as viral vectors (e.g., replication defectiveretroviruses, adenoviruses and adeno-associated viruses), which serveequivalent functions.

The recombinant expression vectors of the invention comprise a nucleicacid of the invention in a form suitable for expression of the nucleicacid in a host cell, which means that the recombinant expression vectorsinclude one or more regulatory sequences, selected on the basis of thehost cells to be used for expression, that is operatively-linked to thenucleic acid sequence to be expressed. Within a recombinant expressionvector, “operably-linked” is intended to mean that the nucleotidesequence of interest is linked to the regulatory sequence(s) in a mannerthat allows for expression of the nucleotide sequence (e.g., in an invitro transcription/translation system or in a host cell when the vectoris introduced into the host cell).

The term “regulatory sequence” is intended to include promoters,enhancers and other expression control elements (e.g., polyadenylationsignals). Such regulatory sequences are described, for example, inGoeddel, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, AcademicPress, San Diego, Calif. (1990). Regulatory sequences include those thatdirect constitutive expression of a nucleotide sequence in many types ofhost cell and those that direct expression of the nucleotide sequenceonly in certain host cells (e.g., tissue-specific regulatory sequences).It will be appreciated by those skilled in the art that the design ofthe expression vector can depend on such factors as the choice of thehost cell to be transformed, the level of expression of protein desired,etc. The expression vectors of the invention can be introduced into hostcells to thereby produce proteins or peptides, including fusion proteinsor peptides, encoded by nucleic acids as described herein. Therecombinant expression vectors of the invention can be designed forexpression of proteins in prokaryotic or eukaryotic cells. For example,proteins can be expressed in bacterial cells such as Escherichia coli,insect cells (using baculovirus expression vectors) yeast cells ormammalian cells. Suitable host cells are discussed further in Goeddel,GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press,San Diego, Calif. (1990). Alternatively, the recombinant expressionvector can be transcribed and translated in vitro, for example using T7promoter regulatory sequences and T7 polymerase.

Expression of proteins in prokaryotes is most often carried out inEscherichia coli with vectors containing constitutive or induciblepromoters directing the expression of either fusion or non-fusionproteins. Fusion vectors add a number of amino acids to a proteinencoded therein, usually to the amino terminus of the recombinantprotein. Such fusion vectors typically serve three purposes: (i) toincrease expression of recombinant protein; (ii) to increase thesolubility of the recombinant protein; and (iii) to aid in thepurification of the recombinant protein by acting as a ligand inaffinity purification. Often, in fusion expression vectors, aproteolytic cleavage site is introduced at the junction of the fusionmoiety and the recombinant protein to enable separation of therecombinant protein from the fusion moiety subsequent to purification ofthe fusion protein. Such enzymes, and their cognate recognitionsequences, include Factor Xa, thrombin and enterokinase. Typical fusionexpression vectors include pGEX (Pharmacia Biotech Inc; Smith andJohnson, 1988. Gene 67: 31-40), pMAL (New England Biolabs, Beverly,Mass.) and pRIT5 (Pharmacia, Piscataway, N.J.) that fuse glutathioneS-transferase (GST), maltose E binding protein, or protein A,respectively, to the target recombinant protein.

Examples of suitable inducible non-fusion E. coli expression vectorsinclude pTrc (Amrann et al., (1988) Gene 69:301-315) and pET 11d(Studier et al., GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185,Academic Press, San Diego, Calif. (1990) 60-89).

One strategy to maximize recombinant protein expression in E. coli is toexpress the protein in a host bacteria with an impaired capacity toproteolytically cleave the recombinant protein. See, e.g., Gottesman,GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press,San Diego, Calif. (1990) 119-128. Another strategy is to alter thenucleic acid sequence of the nucleic acid to be inserted into anexpression vector so that the individual codons for each amino acid arethose preferentially utilized in E. coli (see, e.g., Wada, et al., 1992.Nucl. Acids Res. 20: 2111-2118). Such alteration of nucleic acidsequences of the invention can be carried out by standard DNA synthesistechniques. In another embodiment, the expression vector is a yeastexpression vector. Examples of vectors for expression in yeastSaccharomyces cerivisae include pYepSec (Baldari, et al., 1987. EMBO J.6: 229-234), pMFa (Kurjan and Herskowitz, 1982. Cell 30: 933-943),pJRY88 (Schultz et al., 1987. Gene 54; 113-123), pYES2 (InvitrogenCorporation, San Diego, Calif.), and picZ (InVitrogen Corp, San Diego,Calif.). Alternatively, the polypeptides can be expressed in insectcells using baculovirus expression vectors. Baculovirus vectorsavailable for expression of proteins in cultured insect cells (e.g., SF9cells) include the pAc series (Smith, et al., 1983. Mol. Cell. Biol. 3:2156-2165) and the pVL series (Lucklow and Summers, 1989. Virology 170:31-39).

In yet another embodiment, a nucleic acid of the invention is expressedin mammalian cells using a mammalian expression vector. Examples ofmammalian expression vectors include pCDM8 (Seed, 1987. Nature 329: 840)and pMT2PC (Kaufmnan, et al., 1987. EMBO J. 6: 187-195). When used inmammalian cells, the expression vector's control functions are oftenprovided by viral regulatory elements. For example, commonly usedpromoters are derived from polyoma, adenovirus 2, cytomegalovirus, andsimian virus 40. For other suitable expression systems for bothprokaryotic and eukaryotic cells see, e.g., Chapters 16 and 17 ofSambrook, et al., MOLECULAR CLONING: A LABORATORY MANUAL. 2nd ed., ColdSpring Harbor Laboratory, Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y., 1989.

In another embodiment, the recombinant mammalian expression vector iscapable of directing expression of the nucleic acid preferentially in aparticular cell type (e.g., tissue-specific regulatory elements are usedto express the nucleic acid). Tissue-specific regulatory elements areknown in the art. Non-limiting examples of suitable tissue-specificpromoters include the albumin promoter (liver-specific; Pinkert, et al.,1987. Genes Dev. 1: 268-277), lymphoid-specific promoters (Calame andEaton, 1988. Adv. Immunol. 43: 235-275), in particular promoters of Tcell receptors (Winoto and Baltimore, 1989. EMBO J. 8: 729-733) andimmunoglobulins (Banerji, et al., 1983. Cell 33: 729-740; Queen andBaltimore, 1983. Cell 33: 741-748), neuron-specific promoters (e.g., theneurofilament promoter; Byrne and Ruddle, 1989. Proc. Natl. Acad. Sci.USA 86: 5473-5477), pancreas-specific promoters (Edlund, et al., 1985.Science 230: 912-916), and mammary gland-specific promoters (e.g., milkwhey promoter; U.S. Pat. No. 4,873,316 and European ApplicationPublication No. 264,166). Developmentally-regulated promoters are alsoencompassed, e.g., the murine hox promoters (Kessel and Gruss, 1990.Science 249: 374-379) and the alpha-fetoprotein promoter (Campes andTilghman, 1989. Genes Dev. 3: 537-546).

In one embodiment of the present invention, the starting nucleic acidsegments are first recombined by any of the formats referenced herein togenerate a library of recombinant nucleic acids. The library can vary insize, e.g., ranging from about 10 to about 10⁹ members. In general, theinitial nucleic acid segments, and the recombinant libraries of nucleicacids generated include full-length coding sequences (i.e., open readingframe (ORF), which includes the start codon, coding sequence, and stopcodon), and any essential regulatory sequences, for example, a promoterand polyadenylation sequence, required for expression. However, in theevent that the recombinant nucleic acid does not contain these elements,the recombinant nucleic acids in the library can be inserted into avector that includes the missing sequences prior to screening andselection of recombinant clones.

The recombinant nucleic acid sequences may be combined in an in vivoformat which results in a library of recombinant segments capable ofexpression. Alternatively, the recombination may be performed in vitro,and the recombinant library is introduced into the desired cell typeprior to the step of screening and selection, In some embodiments of theinvention, the recombinant nucleic acid library is amplified in a firsthost, and is then recovered from that host and introduced to a secondhost for reason of expression, selection, or screening, or any otherdesirable parameter. The manner by which the recombinant nucleic acid isintroduced into the host cell depends on the nucleic acid-uptakecharacteristics of the cell type (e.g., having viral receptors, beingcapable of conjugation, being naturally competent, and/or requiringDNA-gun or electropulse). After introduction of the library ofrecombinant DNA genes, the cells may be propagated to allow expressionof genes to occur.

In any of the embodiments, the nucleic acids encoding the lymphokine orlymphokine receptor can be present as: one or more naked DNAs; one ormore nucleic acids disposed in an appropriate expression vector andmaintained episomally; one or more nucleic acids incorporated into thehost cell's genome; a modified version of an endogenous gene encodingthe components of the complex; one or more nucleic acids in combinationwith one or more regulatory nucleic acid sequences; or combinationsthereof. In one embodiment, the host cell's endogenous interleukinand/or interleukin receptor genes are modified using homologousrecombination techniques such that the cell produces a combination ofinterleukin polypeptide, a soluble interleukin receptor polypeptide, andinterleukin/interleukin receptor complex polypeptides, which can beisolated and purified using standard techniques. In any of theembodiments, a nucleic acid encoding the lymphokine component comprisesa member selected from the group consisting of SEQ ID NOs. 1, 2, 14, 15,portions and combinations thereof. In addition, in any of theembodiments, a nucleic acid encoding the lymphokine receptor componentcomprises a member selected from the group consisting of SEQ ID NOs. 3,4, 13, 16, portions and combinations thereof. The nucleic acid encodingthe lymphokine, lymphokine receptor portion, and/orlymphokine/lymphokine receptor chimera may optionally comprise a linkerpeptide or fusion protein component, for example, His-Tag, FLAG-Tag,GFP, GST, an antibody portion, a signal peptide, and the like, at the 5′end, the 3′ end, or at any location within the ORF.

In a preferred embodiment, the nucleic acid of the invention comprises apolynucleotide encoding the soluble (i.e., the extracellular) portion ofa lymphokine receptor. In a particularly preferred embodiment, theinvention comprises a contiguous nucleic acid encoding a signal peptide,a lypmphokine, a linker peptide, and the soluble portion of a lymphokinereceptor, and the Fc portion of an antibody. Any of the embodimentsdescribed herein, can be achieved using standard molecular biologicaland genetic approaches well known to those of ordinary skill in the art.In any of the embodiments a cDNA encoding the open reading frame of SEQID NOs: 1-4, and 13-16 or portions thereof can be incorporated intocommercially available bacterial expression plasmids such as the pGEM(Promega) or pBluescript (Stratagene) vectors, or eukaryotic expressionvectors such as the baculovirus system, pCEP, pcDNA vectors or one oftheir derivatives.

In certain embodiments, the invention comprises an isolatedpolynucleotide sequence encoding the polypeptide of SEQ ID NOs: 5-12 orportions thereof. By “isolated nucleic acid sequence” is meant apolynucleotide that is not immediately contiguous with either of thecoding sequences with which it is immediately contiguous (one on the 5′end and one on the 3′ end) in the naturally occurring genome of theorganism from which it is derived. The term therefore includes, forexample, a recombinant DNA which is incorporated into a vector; into anautomatically replicating plasmid or virus; or into the genomic DNA of aprokaryote or eukaryote, or which exists as a separate molecule (e.g., acDNA) independent of other sequences. The nucleotides can be modifiedforms of DNA or RNA. Modifications include but are not limited to knownsubstitutions of a naturally-occurring base, sugar or internucleoside(backbone) linkage with a modified base such as 5-methylcytosine, amodified sugar such as 2′-methoxy and 2′-fluoro sugars, and modifiedbackbones such as phosphorothioate and methyl phosphonate.

A polynucleotide can be a DNA molecule, a cDNA molecule, genomic DNAmolecule, or an RNA molecule. A polynucleotide as DNA or RNA can includea sequence wherein T (thymidine) can also be U (uracil). Thepolynucleotide can be complementary to SEQ ID NOs: 1-4, and 13-16,wherein complementary refers to the capacity for precise pairing betweentwo nucleotides. For example, if a nucleotide at a certain position of apolynucleotide is capable of forming a Watson-Crick pairing with anucleotide at the same position in an anti-parallel DNA or RNA strand,then the polynucleotide and the DNA or RNA molecule are complementary toeach other at that position. The polynucleotide and the DNA or RNAmolecule are substantially complementary to each other when a sufficientnumber of corresponding positions in each molecule are occupied bynucleotides that can hybridize with each other in order to effect thedesired process. As used herein, hybridization means Watson-Crickhydrogen bonding between complementary nucleoside or nucleotide bases.

In addition, polynucleotides encoding all or a portion of SEQ ID NOs:1-4, and 13-16 are included. Such polynucleotides include naturallyoccurring, synthetic and intentionally manipulated DNA molecules. Forexample, the polynucleotides may be subjected to site-directedmutagenesis by techniques known in the molecular biology art. There are20 naturally occurring amino acids, most of which are specified by morethan one codon. Therefore, degenerate nucleotide sequences are included.The polynucleotides also include polynucleotides coding for polypeptideanalogs, fragments or derivatives of antigenic polypeptides which differfrom naturally-occurring forms in terms of the identity or location ofone or more amino acid residues (deletion analogs containing less thanall of the residues specified for the polypeptide, substitution analogswherein one or more residues specified are replaced by other residuesand addition analogs where in one or more amino acid residues is addedto a terminal or medial portion of the polypeptide) and which share someor all properties of naturally-occurring forms. These molecules includethe incorporation of codons suitable for expression by selectednon-mammalian hosts; the provision of sites for cleavage by restrictionendonuclease enzymes; and the provision of additional initial, terminalor intermediate DNA sequences that facilitate construction of readilyexpressed vectors.

The polynucleotides include polynucleotides that encode polypeptides orfull-length proteins that contain substitutions, insertions, ordeletions into the protein backbone. Related polypeptides are alignedwith by assigning degrees of homology to various deletions,substitutions and other modifications. Homology can be determined alongthe entire polypeptide or polynucleotide or along subsets of contiguousresidues. The percent identity is the percentage of amino acids ornucleotides that are identical when the two sequences are compared. Thepercent similarity is the percentage of amino acids or nucleotides thatare chemically similar when the two sequences are compared. Homologouspolypeptides are preferably greater than or equal to 25%, preferablygreater than or equal to 30%, more preferably greater than or equal to35% or most preferably greater than or equal to 40% identical.

Plasmids disclosed herein are either commercially available, publiclyavailable on an unrestricted basis, or can be constructed from availableplasmids by routine application of well-known, published procedures.Many plasmids and other cloning and expression vectors are well knownand readily available, or those of ordinary skill in the art may readilyconstruct any number of other plasmids suitable for use. These vectorsmay be transformed into a suitable host cell to form a host cell vectorsystem for the production of a polypeptide having the biologicalactivity of a cellular transporter. Suitable hosts include microbes suchas bacteria, yeast, insect or mammalian organisms or cell lines.Examples of suitable bacteria are E. coli and B. subtilis. A preferredyeast vector is pRS426-Gal. Examples of suitable yeast are Saccharomycesand Pichia. Suitable amphibian cells are Xenopus cells. Suitable vectorsfor insect cell lines include baculovirus vectors. Rat or human cellsare preferred mammalian cells.

Transformation of a host cell with recombinant DNA may be carried out byconventional techniques as are well known to those skilled in the art.By “transformation” is meant a permanent or transient genetic changeinduced in a cell following incorporation of new DNA (i.e., DNAexogenous to the cell). Where the cell is a mammalian cell, a permanentgenetic change is generally achieved by introduction of the DNA into thegenome of the cell. By “transformed cell” or “host cell” is meant a cell(e.g., prokaryotic or eukaryotic) into which (or into an ancestor ofwhich) has been introduced, by means of recombinant DNA techniques, aDNA molecule encoding a polypeptide of the invention (i.e., an INDYpolypeptide), or fragment thereof.

Where the host is prokaryotic, such as E. coli, competent cells whichare capable of DNA uptake can be prepared from cells harvested afterexponential growth phase and subsequently treated by the CaCl₂ method byprocedures well known in the art. Alternatively, MgCl₂ or RbCl can beused. Transformation can also be performed after forming a protoplast ofthe host cell or by electroporation.

When the host is a eukaryote, such methods of transfection with DNAinclude calcium phosphate co-precipitates, conventional mechanicalprocedures such as microinjection, electroporation, insertion of aplasmid encased in liposomes, or virus vectors, as well as others knownin the art, may be used. Eukaryotic cells can also be cotransfected withDNA sequences encoding a polypeptide of this disclosure, and a secondforeign DNA molecule encoding a selectable phenotype, such as the herpessimplex thymidine kinase gene. Another method is to use a eukaryoticviral vector, such as simian virus 40 (SV40) or bovine papilloma virus,to transiently infect or transform eukaryotic cells and express theprotein. (Eukaryotic Viral Vectors, Cold Spring Harbor Laboratory,Gluzman ed., 1982). Preferably, a eukaryotic host is utilized as thehost cell as described herein. The eukaryotic cell may be a yeast cell(e.g., Saccharomyces cerevisiae) or may be a mammalian cell, including ahuman cell.

Mammalian cell systems that utilize recombinant viruses or viralelements to direct expression may be engineered. For example, when usingadenovirus expression vectors, the nucleic acid sequences encoding aforeign protein may be ligated to an adenovirustranscription/translation control complex, e.g., the late promoter andtripartite leader sequence. This chimeric gene may then be inserted inthe adenovirus genome by in vitro or in vivo recombination. Insertion ina non-essential region of the viral genome (e.g., region E1 or E3) willresult in a recombinant virus that is viable and capable of expressingthe polypeptides in infected hosts (e.g., Logan & Shenk, Proc. Natl.Acad. Sci. U.S.A. 81:3655-3659, 1984).

For long-term, high-yield production of recombinant proteins, stableexpression is preferred. Rather than using expression vectors thatcontain viral origins of replication, host cells can be transformed withthe cDNA encoding an interleukin/interleukin receptor fusion proteincontrolled by appropriate expression control elements (e.g., promoter,enhancer, sequences, transcription terminators, polyadenylation sites,etc.), and a selectable marker. The selectable marker in the recombinantplasmid confers resistance to the selection and allows cells to stablyintegrate the plasmid into their chromosomes and grow to form foci,which in turn can be cloned and expanded into cell lines. For example,following the introduction of foreign DNA, engineered cells may beallowed to grow for 1 to 2 days in an enriched media, and then areswitched to a selective media. A number of selection systems may beused, including but not limited to the herpes simplex virus thymidinekinase (Wigler et al., Cell 11: 233, 1977), hypoxanthine-guaninephosphoribosyltransferase (Szybalska & Szybalski, Proc. Natl. Sci.U.S.A. 48: 2026, 1962), and adenine phosphoribosyltransferase (Lowy etal., Cell 22: 817, 1980) genes can be employed.

In other embodiments, the invention pertains to isolated nucleic acidmolecules that encode interleukin polypeptides, interleukin receptorpolypeptides, antibody polypeptides, and chimericinterleukin/interleukin receptor polypeptides or biologically activeportions thereof. Also included in the invention are nucleic acidfragments sufficient for use as hybridization probes to identifychimeric interleukin/interleukin receptor-encoding nucleic acids andfragments for use as PCR primers for the amplification and/or mutationof chimeric interleukin/interleukin receptor nucleic acid molecules. Asused herein, the term “nucleic acid molecule” is intended to include DNAmolecules (e.g., cDNA or genomic DNA), RNA molecules (e.g., mRNA),analogs of the DNA or RNA generated using nucleotide analogs, andderivatives, fragments and homologs thereof. The nucleic acid moleculemay be single-stranded or double-stranded, but preferably is compriseddouble-stranded DNA.

In yet another preferred embodiment, the invention includes a method forisolating a nucleic acid of the invention that includes one or more of:(a) recombining nucleic acids from at least two lymphokines orlymphokine receptors to create a library of nucleic acids; (b)transforming the recombinant genes into a competent cell; (c) screeningthe cells; (d) isolating the desired nucleic acid for further cycles ofrecombination with another nucleic acid. The method of this inventionmay also involve the construction of recombinant nucleic acids, plasmidvectors, or both, and the expression of genes in transformed host cells.The molecular cloning techniques required to achieve these goals arewell known in the art.

An interleukin encoding nucleic acid can encode a mature interleukinpolypeptide. As used herein, a “mature” form of a polypeptide or proteindisclosed in the present invention is the product of a naturallyoccurring polypeptide, precursor form, preproprotein or proprotein. Thenaturally occurring polypeptide, precursor or proprotein includes, byway of nonlimiting example, the full-length gene product encoded by thecorresponding gene. Alternatively, it may be defined as the polypeptide,precursor or proprotein encoded by an ORF described herein. The product“mature” form arises, by way of nonlimiting example, as a result of oneor more naturally occurring processing steps that may take place withinthe cell (host cell) in which the gene product arises. Examples of suchprocessing steps leading to a “mature” form of a polypeptide or proteininclude the cleavage of the N-terminal methionine (Met) residue encodedby the initiation codon of an ORF or the proteolytic cleavage of asignal peptide or leader sequence. Thus a mature form arising from aprecursor polypeptide or protein that has residues 1 to n, where residue1 is the N-terminal methionine, would have residues 2 through nremaining after removal of the N-terminal methionine. Alternatively, amature form arising from a precursor polypeptide or protein havingresidues 1 to n, in which an N-terminal signal sequence from residue 1to residue Met is cleaved, would have the residues from residue Met+1 toresidue n remaining. Further as used herein, a “mature” form of apolypeptide or protein may arise from a post-translational modificationother than a proteolytic cleavage event. Such additional processesinclude, by way of non-limiting example, glycosylation, myristoylation,oligomerization or phosphorylation. In general, a mature polypeptide orprotein may result from the operation of only one of these processes, ora combination of any of them.

A nucleic acid of the invention can be amplified using cDNA, mRNA or,alternatively, genomic DNA as a template with appropriateoligonucleotide primers according to standard PCR amplificationtechniques. Furthermore, oligonucleotides corresponding to SEQ ID NOs:1-4, and 13-16 and portions and combinations thereof can be prepared bystandard synthetic techniques, e.g., using an automated DNA synthesizer.

Polypeptides

The present invention is based on the surprising and unexpecteddiscovery that the therapeutic efficacy of interleukins can be enhancedby precoupling or complexing the interleukin to an interleukin receptoror soluble portion thereof. In certain embodiments, the inventionincludes methods for forming the therapeutic polypeptide complex of theinvention. In one embodiment, the method comprises providing a suitableamount of at least one lymphokine polypeptide or portion thereof,providing a suitable amount of at least one lymphokine receptorpolypeptide or portion thereof, admixing the lymphokine and lymphokinereceptor polypeptides under suitable pH and ionic conditions for aduration sufficient to allow complex formation, and optionallyconcentrating or purifying the complex. The polypeptides of the complexcan be formed, for example, using a peptide synthesizer according tostandard methods; by expressing each component polypeptide separately ina cell or cell extract, then isolating and purifying the polypeptide.Optionally, the therapeutic polypeptide complex of the invention can beformed by expressing both polypeptide components of the complex of theinvention in the same cell or cell extract, then isolating and purifyingthe complexes, for example, using chromatographic techniques, such asaffinity chromatography with antibodies to the lymphokine portion, thelymphokine receptor portion, or to the complex. In addition, theinvention includes the expression of a chimera or fusion proteincomprising an interleukin, in-frame and contiguous with an interleukinreceptor or portion thereof.

FIG. 12 demonstrates that the complex of the invention results in atherapeutic composition that exhibits longer half-life in vivo relativeto administration of IL-15 alone. Thus, in a preferred embodiment, thetherapeutic polypeptide complex of the invention comprises at least onelymphokine polypeptide or portion thereof, pre-coupled or complexed withat least one lymphokine receptor, wherein the complex demonstrates an invivo half-life and efficacy greater than IL-15 alone. In anotherembodiment, the complex demonstrates an in vivo half-life of greaterthan about an hour. In one aspect of this embodiment the therapeuticcomplex of the invention is formed from recombinant polypeptidesexpressed in a bacterial or eukaryotic cell or through the use ofchemically synthesized peptides. In certain embodiments, the lymphokinepolypeptide or portion thereof is a member selected from the groupconsisting of SEQ ID NOs. 5, 6, 10, 12, and combinations thereof. Incertain embodiments, the lymphokine receptor polypeptide or portionthereof is a member selected from the group consisting of SEQ ID NOs. 7,8, 9, 11, and combinations thereof. In another embodiment, thetherapeutic complex of the invention demonstrates increased efficacywhen administered to an organism in need thereof, compared to thedelivery of lymphokine, for example, IL-15 or IL-2, alone.

In another of the preferred embodiments, the invention relates to amethod of creating a pre-coupled therapeutic polypeptide complexcomprising at least one interleukin, for example, IL-15, IL-2, portionsor combinations thereof, pre-coupled or complexed with at least oneinterleukin receptor, for example, IL-15Ra, IL-2Ra, portions orcombinations thereof, generated by incubating the interleukinpolypeptide with a soluble interleukin receptor domain or by expressinga novel chimeric nucleic acid molecule comprising the lymphokinepolynucleotide segment and the lymphokine receptor polynucleotidesegment. In a preferred embodiment, the invention provides a method forpre-coupling a lymphokine and a lymphokine receptor comprising providinga lymphokine portion, and a lymphokine receptor portion, and combiningfor a suitable amount of time under ionic and pH buffered conditions toallow complex formation. In a particularly preferred embodiment, thelymphokine polypeptide is selected from the group consisting of SEQ IDNOs. 5, 6, portions or combinations thereof, and the lymphokine receptorpolypeptide is selected from the group consisting of SEQ ID NOs. 7, 8,portions or combinations thereof. In one embodiment, the inventionincludes a method for forming the complex comprising providing theisolated polypeptide components resuspended in a buffer, for examplePBS, admixing the polypeptides, and incubating for from about 1 minuteto about 60 minutes at from about 26° C. to about 40° C. In a furtherembodiment, the lymphokine receptor polypeptide comprises a chimera of alymphokine binding portion and an antibody Fc portion. In a preferredembodiment, SEQ ID NO. 6 or portions thereof, and SEQ ID NO. 8—Fcchimeric molecule are both suspended in PBS, mixed, and incubated forfrom about 20 minutes to about 40 minutes at from about 35° C. to about39° C.

In another embodiment, there is provided substantially pure polypeptideshomologous to SEQ ID NOs: 5-12. A “substantially pure polypeptide” is aninterleukin or interleukin receptor polypeptide, or portion thereof thathas been separated from components that naturally accompany it.Typically, the polypeptide is substantially pure when it is at least60%, by weight, free from the proteins and naturally-occurring organicmolecules with which it is naturally associated. Preferably, thepreparation is at least 75%, more preferably at least 90%, and mostpreferably at least 99%, by weight, interleukin and/or interleukinreceptor polypeptides. A substantially pure polypeptide may be obtained,for example, by extraction from a natural source (e.g., a eukaryoticcell); by expression of a recombinant nucleic acid encoding apolypeptide; or by chemically synthesizing the protein. Purity can bemeasured by any appropriate method, e.g., by column chromatography,polyacrylamide gel electrophoresis, or by HPLC analysis.

Amino acids essential for the function of interleukin and interleukinreceptor polypeptides can be identified according to procedures known inthe art, such as site-directed mutagenesis or alanine-scanningmutagenesis (Cunningham and Wells, Science 244: 1081-1085, 1989; Bass etal., Proc. Natl. Acad. Sci. USA 88: 4498-4502, 1991). In the lattertechnique, single alanine mutations are introduced at different residuesin the molecule, and the resultant mutant molecules are tested forbiological activity (e.g., ligand binding and signal transduction) toidentify amino acid residues that are critical to the activity of themolecule. Sites of ligand-protein interaction can also be determined byanalysis of crystal structure as determined by such techniques asnuclear magnetic resonance, crystallography or photoaffinity labeling.(See, for example, de Vos et al., Science 255: 306-312, 1992; Smith etal., J. Mol. Biol. 224: 899-904, 1992; Wlodaver et al., FEBS Lett. 309:59-64, 1992). The identities of essential amino acids can also beinferred from analysis of homologies with related proteins.

Multiple amino acid substitutions can be made and tested using knownmethods of mutagenesis and screening, such as those disclosed byReidhaar-Olson and Sauer (Science 241: 53-57,1988; or Bowie and Sauer,Proc. Natl. Acad. Sci. USA 86: 2152-2156, 1989). Briefly, these authorsdisclose methods for simultaneously randomizing two or more positions ina polypeptide, selecting for functional polypeptide, and then sequencingthe mutagenized polypeptides to determine the spectrum of allowablesubstitutions at each position. Other methods that can be used includephage display (e.g., Lowman et al., Biochem. 30: 10832-10837, 1991;Ladner et al., U.S. Pat. No. 5,223,409; Huse, WIPO Publication WO92/06204) and region-directed mutagenesis (Derbyshire et al., Gene 46:145, 1986; Ner et al., DNA 7: 127, 1988). Mutagenesis methods asdisclosed above can be combined with high-throughput screening methodsto detect the activity of cloned, mutagenized proteins in host cells.Mutagenized DNA molecules that encode active proteins or portionsthereof (e.g., ligand-binding fragments) can be recovered from the hostcells and rapidly sequenced using modern equipment. These methods allowthe rapid determination of the importance of individual amino acidresidues in a polypeptide of interest, and can be applied topolypeptides of unknown structure.

Using the methods discussed above, one of ordinary skill in the art canprepare a variety of polypeptides that are substantially homologous toSEQ ID NO: 5-12 or allelic variants thereof and retain the properties ofthe wild-type polypeptides. As expressed and claimed herein thelanguage, “a polypeptide as defined by SEQ ID NO: 5-12” includes allallelic variants and species orthologs of the polypeptides. The term“polypeptide” as used herein includes modified sequences such asglycoproteins, and is specifically intended to cover naturally occurringpolypeptides or proteins, as well as those that are recombinantly orsynthetically synthesized, which occur in at least two differentconformations wherein both conformations have the same or substantiallythe same amino acid sequence but have different three dimensionalstructures. “Fragments” are a portion of a naturally occurring protein.Fragments can have the same or substantially the same amino acidsequence as the naturally occurring protein.

The disclosure also encompasses proteins that are functionallyequivalent to the interleukin and interleukin receptor gene product, asjudged by any of a number of criteria, including but not limited to theresulting biological effect, for example, a change in phenotype such asproliferation of immune cells, changes in gene expression, for example,specific biomarkers which confirm activation of the IL-15 and/or IL-2signaling pathways. Such functionally equivalent proteins includeadditions or substitutions of amino acid residues within the amino acidsequence encoded by the nucleotide sequences described, but which resultin a silent change or “conservative mutation”, thus producing afunctionally equivalent gene product. In the case of polypeptidesequences that are less than 100% identical to a reference sequence, thenon-identical positions are preferably, but not necessarily,conservative substitutions for the reference sequence. Preferably,conservative amino acid substitutions are made at one or more predicted,non-essential amino acid residues. A “conservative amino acidsubstitution” is one in which the amino acid residue is replaced with anamino acid residue having a similar side chain. Families of amino acidresidues having similar side chains have been defined within the art.These families include amino acids with basic side chains (e.g., lysine,arginine, histidine), acidic side chains (e.g., aspartic acid, glutamicacid), uncharged polar side chains (e.g., glycine, asparagine,glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains(e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine,methionine, tryptophan), beta-branched side chains (e.g., threonine,valine, isoleucine) and aromatic side chains (e.g., tyrosine,phenylalanine, tryptophan, histidine). Thus, a predicted non-essentialamino acid residue in the protein is replaced with another amino acidresidue from the same side chain family. Alternatively, in anotherembodiment, mutations can be introduced randomly along all or part ofthe coding sequence, such as by saturation mutagenesis, and theresultant mutants can be screened for biological activity to identifymutants that retain activity. Following mutagenesis, the encoded proteincan be expressed by any recombinant technology known in the art and theactivity of the protein can be determined.

The polynucleotides can also be designed to provide additionalsequences, such as, for example, the addition of coding sequences foradded C-terminal or N-terminal amino acids that would facilitatepurification by trapping on columns or use of antibodies. Such tagsinclude, for example, histidine-rich tags that allow purification ofpolypeptides on Nickel columns. Such gene modification techniques andsuitable additional sequences are well known in the molecular biologyarts.

In another embodiment, the invention relates to a peptide complexcomprising a polypeptide having at least 30% homology to a memberselected from the group consisting of SEQ ID NOs. 5, 6, 10, 12, portionsand combinations thereof, with a polypeptide having at least 30%homology to a member selected from the group consisting of SEQ ID NOs.7, 8, 9, 11, portions and combinations thereof. In another embodiment,the invention relates to a peptide complex comprising a polypeptidehaving at least 80% homology to a member selected from the groupconsisting of SEQ ID NOs. 5, 6, 10, 12, portions and combinationsthereof, with a polypeptide having at least 80% homology to a memberselected from the group consisting of SEQ ID NOs. 7, 8, 9, 11, portionsand combinations thereof. In another embodiment, the invention comprisesa member selected from the group consisting of SEQ ID NOs. 5, 6, 10, 12,portions and combinations thereof, coupled or complexed with a memberselected from the group consisting of SEQ ID NOs. 7, 8, 9, 11, portionsand combinations thereof.

Cellular proliferation, for example, immune cell proliferation, decreasein tumor burden or formation or increase in tumor resistance, areparameters that can be used to evaluate the efficacy of the complex ofthe invention. It will be understood by one skilled in the art thatthere are many methods for evaluating the proliferative capacity ofcells that are suitable for use in the methods of the invention. Forexample, cells can be labeled in vitro (or in vivo) with BrdU todetermine the percent of dividing cells or evaluated using a colonyforming assay, as described in Li et al. (1997), supra. Cell suitablefor the analysis of proliferative capacity include cells grown in tissueculture, cells isolated from an animal that has been treated with a testcompound, cells that are part of a live animal, or cells that are partof a tissue section obtained from an animal.

In more than one embodiment of the above assay methods of the invention,it may be desirable to immobilize either the interleukin, interleukinreceptor, or interleukin/interleukin receptor complex to facilitateseparation of complexed from uncomplexed forms the proteins. In oneembodiment, a interleukin or interleukin receptor fusion protein can beprovided that adds a domain that allows one or both of the proteins tobe bound to a matrix. For example, GST-fusion proteins or GST-targetfusion proteins can be adsorbed onto glutathione sepharose beads (SigmaChemical, St. Louis, Mo.) or glutathione derivatized microtiter plates,and the mixture of interleukin and interleukin receptor is incubatedunder conditions conducive to complex formation (e.g., at physiologicalconditions for salt and pH). Following incubation, the beads ormicrotiter plate wells are washed to remove any unbound components, thematrix immobilized in the case of beads, and amount of complexdetermined either directly or indirectly. Alternatively, the complexescan be dissociated from the matrix, and the amount or activitydetermined using standard techniques.

Other techniques for immobilizing proteins on matrices can also be usedin the invention. For example, either the proteins can be immobilizedutilizing conjugation of biotin and streptavidin. Biotinylated proteinmolecules can be prepared from biotin-NHS (N-hydroxy-succinimide) usingtechniques well-known within the art (e.g., biotinylation kit, PierceChemicals, Rockford, Ill.), and immobilized in the wells ofstreptavidin-coated 96 well plates (Pierce Chemical). Alternatively,antibodies reactive with the interleukin or interleukin receptorpolypeptides, but which do not interfere with binding, can bederivatized to the wells of the plate, and protein complexes trapped inthe wells by antibody conjugation. Methods for detecting such complexes,in addition to those described above for the GST-immobilized complexes,include immunodetection of complexes using antibodies reactive with theinterleukin or interleukin receptor polypeptides, as well asenzyme-linked assays that rely on detecting an enzymatic activityassociated with the interleukin or interleukin receptor polypeptides.

Another preferred embodiment relates to methods for modulating,inhibiting or augmenting an immune response comprising administering aneffective amount of the therapeutic polypeptide complex of the inventionto an individual. Other aspects of this method include theadministration of an effective amount of the therapeutic polypeptidecomplex in combination with at least one pharmaceutically acceptableexcipient, adjuvant, biologically active agent or a combination thereof.

In another embodiment the invention provides a method for augmenting theimmunity of an organism in need thereof by the administration of apre-coupled complex of a lymphokine and lymphokine receptor thatdemonstrates a substantially longer in vivo half-life, and substantiallygreater efficacy than the lymphokine alone. In addition, this embodimentalso includes a method of driving homeostatic proliferation oflymphokine responsive immune cells, for example, T cells, B cells, NKcells or the like. In certain of the preferred embodiments, the presentembodiment includes the use of a lymphokine receptor molecule whichcontains a human immunoglobulin Fc fragment. For example, an IL-15Ra-Fcconstruct is commercially available from R&D Systems (Minneapolis,Minn.). Additionally, it will be recognized by one of ordinary skill inthe art that the lymphokine receptor domain of the complex of theinvention may optionally have the immunoglobulin Fc fragment removed.

In another embodiment, the present invention includes a method forapplying the complex as an adjuvant to increase immune responses tocancer, infection, or to augment vaccination of any kind. In one aspectof this embodiment the complex of the invention is used to enhanceimmune system reconstitution following bone marrow, stem celltransplantation or in cases of immunodeficiency such as AIDS. Thepresent invention also includes a method of using the complex of theinvention to assist in the growth of lymphocytes in vitro which may thenbe used for adoptive immunotherapy comprising providing a patient;removing a volume of blood from the patient and isolating the patient'slymphocytes; treating the lymphocytes with an effective amount of thecomplex of the invention; and administering the treated lymphocytes backinto the patient.

In still another of the preferred embodiments, the invention includes amethod of using the complex modified to be used as an antagonist oflymphokine activity, for example, IL-15. For example, through sequencemodifications of a lymphokine or lymphokine receptor, for example, IL-15or IL-15Ra, the combined complex could be rendered an inhibitor of thelymphokine activity in vivo. Such a molecule could have potentialtherapeutic effects in inhibiting autoimmunity, transplant rejection orgraft-versus host disease.

In any of the preferred embodiments, any of the possible recombinantforms of the lymphokine/lymphokine receptor complex molecule arecontemplated. For example, a single chain polypeptide molecule producedfrom a genetic construct containing the lymphokine gene, for example,IL-2, IL-15, portions or combinations thereof, fused to the lymphokinereceptor gene, for example, IL-2Ra, IL-15Ra, portions or combinationsthereof, and optionally the Fc portion of an antibody. In certainembodiments the genetic construct can further include one or morenucleic acid sequences that encode a linker polypeptides. In addition toserving to relieve steric or conformational restraints in the complex,it is conceivable that the linker sequence could impart other qualities,for example, a nuclease recognition sequence, a protease recognitionsequence, a photo-reactive domain, a hydrophobic domain, hydrophilicdomain, an active domain, enzymatic function, a site for chemicalmodification or conjugation, purification or the like. In furtherembodiments, the invention provides chimeric molecules comprised of atleast one lymphokine gene and at least one lymphokine receptor geneligated in tandem such that would allow expression of multimeric formsof the complex, for example, dimers, trimers, and the like. Theseproteins could also be produced in eukaryotic or prokaryotic cells.

Chimeric and Fusion Proteins

As described supra, the invention also provides chimeric or fusionproteins. As used herein, A “chimeric protein” or “fusion protein”comprises a polypeptide operatively-linked to another polypeptide, forexample, one or more of the polypeptides chosen from SEQ ID NOs: 5-12,or portions thereof. Whereas the polypeptides chosen from SEQ ID NOs:5-12 include polypeptides having an amino acid sequence with at least30% homology. Within the fusion protein the polypeptide can correspondto all or a portion of a polypeptide chosen from SEQ ID NOs: 5-12. Inone embodiment, the fusion protein comprises at least one biologicallyactive portion of the protein. In another embodiment, the fusion proteincomprises at least two biologically active portions of at least oneprotein chosen from SEQ ID NOs: 5-12. In yet another embodiment, thefusion protein comprises at least three biologically active portions ofat least one protein chosen from SEQ ID NOs: 5-12. Within the fusionprotein, the term “operatively-linked” is intended to indicate that thediscrete polypeptides are fused in-frame with one another at theN-terminus or C-terminus.

In more than one embodiment of the above assay methods, it may bedesirable to immobilize the chimeric polypeptides of the invention tofacilitate separation of the proteins. In one embodiment, a fusionprotein can be provided which adds a domain that allows the proteins tobe bound to a matrix. For example, glutathione-S-transferase fusionproteins or conjugation of biotin and streptavidin.

In one embodiment, the fusion protein is a GST-fusion protein in whichthe polypeptide sequences are fused to the C-terminus or N-terminus ofthe GST (glutathione S-transferase) sequences. In another embodiment,the fusion protein contains a heterologous signal sequence at itsN-terminus. In certain host cells (e.g., mammalian host cells),expression and/or secretion can be increased through use of aheterologous signal sequence. In yet another embodiment, the fusionprotein is immunoglobulin fusion protein in which the polypeptides orpolypeptide complex of the invention is fused to sequences derived froma member of the immunoglobulin protein family. In one embodiment, theimmunoglobulin fusion proteins of the invention can be incorporated intopharmaceutical compositions and administered to a subject to modulate aninteraction between a ligand and a protein on the surface of a cell. Theimmunoglobulin fusion proteins can be used to affect the bioavailabilityof a cognate ligand. Inhibition of the ligand interaction may be usefultherapeutically for both the treatment of proliferative anddifferentiative disorders, as well as modulating (e.g. promoting orinhibiting) cell survival. Moreover, the immunoglobulin fusion proteinsof the invention can be used as immunogens to produce antibodies in asubject, to purify ligands, and in screening assays to identifymolecules that inhibit the interaction.

A chimeric or fusion protein of the invention can be produced bystandard recombinant DNA techniques. For example, DNA fragments codingfor the different polypeptide sequences are ligated together in-frame inaccordance with conventional techniques, e.g., by employing blunt-endedor stagger-ended termini for ligation, restriction enzyme digestion toprovide for appropriate termini, filling-in of cohesive ends asappropriate, alkaline phosphatase treatment to avoid undesirablejoining, and enzymatic ligation. In another embodiment, the fusion genecan be synthesized by conventional techniques including automated DNAsynthesizers. Alternatively, PCR amplification of gene fragments can becarried out using anchor primers that give rise to complementaryoverhangs between two consecutive gene fragments that can subsequentlybe annealed and reamplified to generate a chimeric gene sequence (see,e.g., Ausubel, et al. (eds.) CURRENT PROTOCOLS IN MOLECULAR BIOLOGY,John Wiley & Sons, 1992). Moreover, many expression vectors arecommercially available that already encode a fusion moiety (e.g., a GSTpolypeptide). One or more of SEQ ID NOs: 1-4, and 13-16 can be clonedinto such an expression vector such that the fusion moiety is linkedin-frame to the desired polypeptide.

Antibodies

The term “antibody” as used herein refers to immunoglobulin moleculesand immunologically active portions of immunoglobulin (Ig) molecules,i.e., molecules that contain an antigen-binding site that specificallybinds (immunoreacts with) an antigen, comprising at least one, andpreferably two, heavy (H) chain variable regions (abbreviated herein asVH), and at least one and preferably two light (L) chain variableregions (abbreviated herein as VL). Such antibodies include, but are notlimited to, polyclonal, monoclonal, chimeric, single chain, Fab, Fab′and F(ab′)2 fragments, and an Fab expression library. The VH and VLregions can be further subdivided into regions of hypervariability,termed “complementarity determining regions” (“CDR”), interspersed withregions that are more conserved, termed “framework regions” (FR). Theextent of the framework region and CDR's has been precisely defined(see, Kabal, E. A., et al. (1991) Sequences of Proteins of ImmunologicalInterest, Fifth Edition, U.S. Department of Health and Human Services,NIH Publication No. 91-3242, and Chothia, C. et al. (1987) J. Mol. Biol.196:901-917, which are incorporated herein by reference). Each VH and VLis composed of three CDR's and four FRs, arranged from amino-terminus tocarboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3,CDR3, FR4. In general, antibody molecules obtained from humans relatesto any of the classes IgG, IgM, IgA, IgE and IgD, which differ from oneanother by the nature of the heavy chain present in the molecule.Certain classes have subclasses as well, such as IgG₁, IgG₂, and others.Furthermore, in humans, the light chain may be a kappa chain or a lambdachain. Reference herein to antibodies includes a reference to all suchclasses, subclasses and types of human antibody species.

Antibodies can be prepared from the intact polypeptide or fragmentscontaining peptides of interest as the immunizing agent. A preferredantigenic polypeptide fragment is 15-100 contiguous amino acids of SEQID NOs: 5-12. In one embodiment, the peptide is located in anon-transmembrane domain of the polypeptide, e.g., in an extracellularor intracellular domain. An exemplary antibody or antibody fragmentbinds to an epitope that is accessible from the extracellular milieu andthat alters the functionality of the protein. In certain embodiments,the present invention comprises antibodies that recognize and arespecific for one or more epitopes of any of SEQ ID NOs: 5-12, variants,portions and/or combinations thereof. In other embodiments, theantibodies of the invention may be specific for theinterleukin/interleukin receptor complex itself. In still otherembodiments an antibody specific for an interleukin may function as the“interleukin receptor”—i.e., functioning in a transpresentationmechanism similar to that observed with a complex involving the solubleportion of the interleukin receptor polypeptides, i.e., IL-15Ra and/orIL-2Ra. In alternative embodiments antibodies of the invention maytarget and interfere with the interleukin/interleukin receptorinteraction to inhibit interleukin signaling.

The preparation of polyclonal antibodies is well known in the molecularbiology art; see for example, Production of Polyclonal Antisera inImmunochemical Processes (Manson, ed.), pages 1-5 (Humana Press 1992)and Coligan et al., Production of Polyclonal Antisera in Rabbits, Rats,Mice and Hamsters in Current Protocols in Immunology, section 2,4.1(1992), The preparation of monoclonal antibodies is also well known inthe art; see for example, Harlow et al., Antibodies: A LaboratoryManual, page 726 (Cold Spring Harbor Pub. 1988).

Monoclonal antibodies can be obtained by injecting mice or rabbits witha composition comprising an antigen, verifying the presence of antibodyproduction by removing a serum sample, removing the spleen to obtain Blymphocytes, fusing the lymphocytes with myeloma cells to producehybridomas, cloning the hybridomas, selecting positive clones thatproduce antibodies to the antigen, and isolating the antibodies from thehybridoma cultures. Monoclonal antibodies can be isolated and purifiedfrom hybridoma cultures by techniques well known in the art.

In other embodiments, the antibody can be recombinantly produced, e.g.,produced by phage display or by combinatorial methods. Phage display andcombinatorial methods can be used to isolate recombinant antibodies thatbind to SEQ ID NOs: 5-12 or fragments thereof (as described in, e.g.,Ladner et al. U.S. Pat. No. 5,223,409; Kang et al. InternationalPublication No. WO 92/18619; Dower et al. International Publication No.WO 91/17271; Winter et al. International Publication WO 92/20791;Markland et al. International Publication No. WO 92/15679; Breitling etal. International Publication WO 93/01288; McCafferty et al.International Publication No. WO 92/01047; Garrard et al. InternationalPublication No. WO 92/09690; Ladner et al. International Publication No.WO 90/02809; Fuchs et al. (1991) Bio/Technology 9:1370-1372; Hay et al.(1992) Hum Antibod Hybridomas 3:81-85; Huse et al. (1989) Science246:1275-1281; Griffths et al. (1993) EMBO J. 12:725-734; Hawkins et al.(1992) J Mol Biol 226:889-896; Clackson et al. (1991) Nature352:624-628; Gram et al. (1992) PNAS 89:3576-3580; Garrad et al. (1991)Bio/Technology 9:1373-1377; Hoogenboom et al. (1991) Nuc Acid Res19:4133-4137; and Barbas et al. (1991) PNAS 88:7978-7982).

Human monoclonal antibodies can also be generated using transgenic micecarrying the human immunoglobulin genes rather than the mouse system.Splenocytes from these transgenic mice immunized with the antigen ofinterest are used to produce hybridomas that secrete human mAbs withspecific affinities for epitopes from a human protein (see, e.g., Woodet al. International Application WO 91/00906, Kucherlapati et al. PCTpublication WO 91/10741; Lonberg et al. International Application WO92/03918; Kay et al. International Application 92/03917; Lonberg, N. etal. 1994 Nature 368:856-859; Green, L. L. et al. 1994 Nature Genet.7:13-21; Morrison, S. L. et al. 1994 Proc. Natl. Acad. Sci. USA81:6851-6855; Bruggeman et al. 1993 Year Immunol 7:33-40; Tuaillon etal. 1993 PNAS 90:3720-3724; Bruggeman et al. 1991 Eur J Immunol21:1323-1326).

A therapeutically useful antibody to the components of the complex ofthe invention or the complex itself may be derived from a “humanized”monoclonal antibody. Humanized monoclonal antibodies are produced bytransferring mouse complementarity determining regions from heavy andlight variable chains of the mouse immunoglobulin into a human variabledomain, then substituting human residues into the framework regions ofthe murine counterparts. The use of antibody components derived fromhumanized monoclonal antibodies obviates potential problems associatedwith immunogenicity of murine constant regions. Techniques for producinghumanized monoclonal antibodies can be found in Jones et al., Nature321: 522, 1986 and Singer et al., J. Immunol. 150: 2844, 1993. Theantibodies can also be derived from human antibody fragments isolatedfrom a combinatorial immunoglobulin library; see, for example, Barbas etal., Methods: A Companion to Methods in Enzymology 2, 119, 1991.

In addition, chimeric antibodies can be obtained by splicing the genesfrom a mouse antibody molecule with appropriate antigen specificitytogether with genes from a human antibody molecule of appropriatebiological specificity; see, for example, Takeda et al., Nature 314:544-546, 1985. A chimeric antibody is one in which different portionsare derived from different animal species.

Anti-idiotype technology can be used to produce monoclonal antibodiesthat mimic an epitope. An anti-idiotypic monoclonal antibody made to afirst monoclonal antibody will have a binding domain in thehypervariable region that is the “image” of the epitope bound by thefirst monoclonal antibody. Alternatively, techniques used to producesingle chain antibodies can be used to produce single chain antibodies.Single chain antibodies are formed by linking the heavy and light chainfragments of the Fv region via an amino acid bridge, resulting in asingle chain polypeptide. Antibody fragments that recognize specificepitopes, e.g., extracellular epitopes, can be generated by techniqueswell known in the art. Such fragments include Fab fragments produced byproteolytic digestion, and Fab fragments generated by reducing disulfidebridges. When used for immunotherapy, the monoclonal antibodies,fragments thereof, or both may be unlabelled or labeled with atherapeutic agent. These agents can be coupled directly or indirectly tothe monoclonal antibody by techniques well known in the art, and includesuch agents as drugs, radioisotopes, lectins and toxins.

The dosage ranges for the administration of monoclonal antibodies arelarge enough to produce the desired effect, and will vary with age,condition, weight, sex, age and the extent of the condition to betreated, and can readily be determined by one skilled in the art.Dosages can be about 0.1 mg/kg to about 2000 mg/kg. The monoclonalantibodies can be administered intravenously, intraperitoneally,intramuscularly, and/or subcutaneously.

In certain embodiments of the invention, at least one epitopeencompassed by the antigenic peptide is a region of SEQ ID NOs: 5-12that is located on the surface of the protein, e.g., a hydrophilicregion. A hydrophobicity analysis of the protein sequence will indicatewhich regions of a polypeptide are particularly hydrophilic and,therefore, are likely to encode surface residues useful for targetingantibody production. As a means for targeting antibody production,hydropathy plots showing regions of hydrophilicity and hydrophobicitymay be generated by any method well known in the art, including, forexample, the Kyte Doolittle or the Hopp Woods methods, either with orwithout Fourier transformation. See, e.g., Hopp and Woods, 1981, Proc.Nat. Acad. Sci. USA 78: 3824-3828; Kyte and Doolittle 1982, J. Mol.Biol. 157: 105-142, each incorporated herein by reference in theirentirety. Antibodies that are specific for one or more domains within anantigenic protein, or derivatives, fragments, analogs or homologsthereof, are also provided herein. A protein of the invention, or aderivative, fragment, analog, homolog or ortholog thereof, may beutilized as an immunogen in the generation of antibodies thatimmunospecifically bind these protein components.

Human Antibodies

Fully human antibodies essentially relate to antibody molecules in whichthe entire sequence of both the light chain and the heavy chain,including the CDRs, arise from human genes. Such antibodies are termed“human antibodies”, or “fully human antibodies” herein. Human monoclonalantibodies can be prepared by the trioma technique; the human B-cellhybridoma technique (see Kozbor, et al., 1983 Immunol Today 4: 72) andthe EBV hybridoma technique to produce human monoclonal antibodies (seeCole, et al., 1985 In: MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R.Liss, Inc., pp. 77-96). Human monoclonal antibodies may be utilized inthe practice of the present invention and may be produced by using humanhybridomas (see Cote, et al., 1983. Proc Natl Acad Sci USA 80:2026-2030) or by transforming human B-cells with Epstein Barr Virus invitro (see Cole, et al., 1985 In: MONOCLONAL ANTIBODIES AND CANCERTHERAPY, Alan R. Liss, Inc., pp. 77-96).

In addition, human antibodies can also be produced using additionaltechniques, including phage display libraries (Hoogenboom and Winter, J.Mol. Biol. 227:381 (1991); Marks et al., J. Mol. Biol., 222:581 (1991)).Similarly, human antibodies can be made by introducing humanimmunoglobulin loci into transgenic animals, e.g., mice in which theendogenous immunoglobulin genes have been partially or completelyinactivated. Upon challenge, human antibody production is observed,which closely resembles that seen in humans in all respects, includinggene rearrangement, assembly, and antibody repertoire. This approach isdescribed, for example, in U.S. Pat. Nos. 5,545,807; 5,545,806;5,569,825; 5,625,126; 5,633,425; 5,661,016, and in Marks et al.(Bio/Technology 10, 779-783 (1992)); Lonberg et al. (Nature 368 856-859(1994)); Morrison (Nature 368, 812-13 (1994)); Fishwild et al, (NatureBiotechnology 14, 845-51 (1996)); Neuberger (Nature Biotechnology 14,826 (1996)); and Lonberg and Huszar (Intern. Rev. Immunol. 13 65-93(1995)).

Human antibodies may additionally be produced using transgenic nonhumananimals which are modified so as to produce fully human antibodiesrather than the animal's endogenous antibodies in response to challengeby an antigen. (See PCT publication WO94/02602). The endogenous genesencoding the heavy and light immunoglobulin chains in the nonhuman hosthave been incapacitated, and active loci encoding human heavy and lightchain immunoglobulins are inserted into the host's genome. The humangenes are incorporated, for example, using yeast artificial chromosomescontaining the requisite human DNA segments. An animal which providesall the desired modifications is then obtained as progeny bycrossbreeding intermediate transgenic animals containing fewer than thefull complement of the modifications. The preferred embodiment of such anonhuman animal is a mouse, and is termed the Xenomouse™ as disclosed inPCT publications WO 96/33735 and WO 96/34096. This animal produces Bcells which secrete fully human immunoglobulins. The antibodies can beobtained directly from the animal after immunization with an immunogenof interest, as, for example, a preparation of a polyclonal antibody, oralternatively from immortalized B cells derived from the animal, such ashybridomas producing monoclonal antibodies. Additionally, the genesencoding the immunoglobulins with human variable regions can berecovered and expressed to obtain the antibodies directly, or can befurther modified to obtain analogs of antibodies such as, for example,single chain Fv molecules.

Fab Fragments and Single Chain Antibodies

According to the invention, techniques can be adapted for the productionof single-chain antibodies specific to an antigenic protein of theinvention (see e.g., U.S. Pat. No. 4,946,778). In addition, methods canbe adapted for the construction of Fab expression libraries (see e.g.,Huse, et al., 1989 Science 246: 1275-1281) to allow rapid and effectiveidentification of monoclonal Fab fragments with the desired specificityfor a protein or derivatives, fragments, analogs or homologs thereof.Antibody fragments that contain the idiotypes to a protein antigen maybe produced by techniques known in the art including, but not limitedto: (i) an F(ab′)2 fragment produced by pepsin digestion of an antibodymolecule; (ii) an Fab fragment generated by reducing the disulfidebridges of an F(ab′)2 fragment; (iii) an Fab fragment generated by thetreatment of the antibody molecule with papain and a reducing agent and(iv) Fv fragments.

Bispecific Antibodies

Bispecific antibodies are monoclonal, preferably human or humanized,antibodies that have binding specificities for at least two differentantigens. In the present case, one of the binding specificities is foran antigenic protein of the invention. The second binding target is anyother antigen, and advantageously is a cell-surface protein or receptoror receptor subunit.

Methods for making bispecific antibodies are known in the art.Traditionally, the recombinant production of bispecific antibodies isbased on the co-expression of two immunoglobulin heavy-chain/light-chainpairs, where the two heavy chains have different specificities (Milsteinand Cuello, Nature, 305:537-539 (1983)). Because of the randomassortment of immunoglobulin heavy and light chains, these hybridomas(quadromas) produce a potential mixture of ten different antibodymolecules, of which only one has the correct bispecific structure. Thepurification of the correct molecule is usually accomplished by affinitychromatography steps. Similar procedures are disclosed in WO 93/08829,published May 13, 1993, and in Traunecker et al., EMBO J., 10:3655-3659(1991).

Antibody variable domains with the desired binding specificities(antibody-antigen combining sites) can be fused to immunoglobulinconstant domain sequences. The fusion preferably is with animmunoglobulin heavy-chain constant domain, comprising at least part ofthe hinge, CH2, and CH3 regions. It is preferred to have the firstheavy-chain constant region (CH1) containing the site necessary forlight-chain binding present in at least one of the fusions. DNAsencoding the immunoglobulin heavy-chain fusions and, if desired, theimmunoglobulin light chain, are inserted into separate expressionvectors, and are co-transfected into a suitable host organism. Forfurther details of generating bispecific antibodies see, for example,Suresh et al., Methods in Enzymology, 121:210 (1986).

According to another approach described in WO 96/27011, the interfacebetween a pair of antibody molecules can be engineered to maximize thepercentage of heterodimers which are recovered from recombinant cellculture. The preferred interface comprises at least a part of the CH3region of an antibody constant domain. In this method, one or more smallamino acid side chains from the interface of the first antibody moleculeare replaced with larger side chains (e.g. tyrosine or tryptophan).Compensatory “cavities” of identical or similar size to the large sidechain(s) are created on the interface of the second antibody molecule byreplacing large amino acid side chains with smaller ones (e.g. alanineor threonine). This provides a mechanism for increasing the yield of theheterodimer over other unwanted end-products such as homodimers.

Bispecific antibodies can be prepared as full length antibodies orantibody fragments (e.g. F(ab′)2 bispecific antibodies). Techniques forgenerating bispecific antibodies from antibody fragments have beendescribed in the literature. For example, bispecific antibodies can beprepared using chemical linkage. Brennan et al., Science 229:81 (1985)describe a procedure wherein intact antibodies are proteolyticallycleaved to generate F(ab′)2 fragments. These fragments are reduced inthe presence of the dithiol complexing agent sodium arsenite tostabilize vicinal dithiols and prevent intermolecular disulfideformation. The Fab′ fragments generated are then converted tothionitrobenzoate (TNB) derivatives. One of the Fab′-TNB derivatives isthen reconverted to the Fab′-thiol by reduction with mercaptoethylamineand is mixed with an equimolar amount of the other Fab′-TNB derivativeto form the bispecific antibody. The bispecific antibodies produced canbe used as agents for the selective immobilization of enzymes.

Additionally, Fab′ fragments can be directly recovered from E. coli andchemically coupled to form bispecific antibodies. Shalaby et al., J.Exp. Med. 175:217-225 (1992) describe the production of a fullyhumanized bispecific antibody F(ab′)2 molecule. Each Fab′ fragment wasseparately secreted from E. coli and subjected to directed chemicalcoupling in vitro to form the bispecific antibody. The bispecificantibody thus formed was able to bind to cells overexpressing the ErbB2receptor and normal human T cells, as well as trigger the lytic activityof human cytotoxic lymphocytes against human breast tumor targets.

Various techniques for making and isolating bispecific antibodyfragments directly from recombinant cell culture have also beendescribed. For example, bispecific antibodies have been produced usingleucine zippers. Kostelny et al., J. Immunol. 148(5):1547-1553 (1992).The leucine zipper peptides from the Fos and Jun proteins were linked tothe Fab′ portions of two different antibodies by gene fusion. Theantibody homodimers were reduced at the hinge region to form monomersand then re-oxidized to form the antibody heterodimers. This method canalso be utilized for the production of antibody homodimers. The“diabody” technology described by Hollinger et al., Proc. Natl. Acad.Sci. USA 90:6444-6448 (1993) has provided an alternative mechanism formaking bispecific antibody fragments. The fragments comprise aheavy-chain variable domain (VH) connected to a light-chain variabledomain (VL) by a linker which is too short to allow pairing between thetwo domains on the same chain. Accordingly, the VH and VL domains of onefragment are forced to pair with the complementary VL and VH domains ofanother fragment, thereby forming two antigen-binding sites. Anotherstrategy for making bispecific antibody fragments by the use ofsingle-chain Fv (sFv) dimers has also been reported. See, Gruber et al.,J. Immunol. 152:5368 (1994). Antibodies with more than two valencies arecontemplated. For example, trispecific antibodies can be prepared. Tuttet al., J. Immunol. 147:60 (1991).

Exemplary bispecific antibodies can bind to two different epitopes, atleast one of which originates in the protein antigen of the invention.Alternatively, an anti-antigenic arm of an immunoglobulin molecule canbe combined with an arm which binds to a triggering molecule on aleukocyte such as a T-cell receptor molecule (e.g. CD2, CD3, CD28, orB7), or Fc receptors for IgG (FcgammaR), such as FcgammaRI (CD64),FcgammaRII (CD32) and FcgammaRIII (CD16) so as to focus cellular defensemechanisms to the cell expressing the particular antigen. Bispecificantibodies can also be used to direct cytotoxic agents to cells whichexpress a particular antigen. These antibodies possess anantigen-binding arm and an arm which binds a cytotoxic agent or aradionuclide chelator, such as EOTUBE, DPTA, DOTA, or TETA.

Heteroconjugate Antibodies

Heteroconjugate antibodies are also within the scope of the presentinvention. Heteroconjugate antibodies are composed of two covalentlyjoined antibodies. Such antibodies have, for example, been proposed totarget immune system cells to unwanted cells (U.S. Pat. No. 4,676,980),and for treatment of HIV infection (WO 91/00360; WO 92/200373; EP03089). It is contemplated that the antibodies can be prepared in vitrousing known methods in synthetic protein chemistry, including thoseinvolving crosslinking agents. For example, immunotoxins can beconstructed using a disulfide exchange reaction or by forming athioether bond. Examples of suitable reagents for this purpose includeiminothiolate and methyl-4-mercaptobutyrimidate and those disclosed, forexample, in U.S. Pat. No. 4,676,980.

Immunoconjugates

The invention also pertains to immunoconjugates comprising an antibodyconjugated to a cytotoxic agent such as a chemotherapeutic agent, toxin(e.g., an enzymatically active toxin of bacterial, fungal, plant, oranimal origin, or fragments thereof), or a radioactive isotope (i.e., aradioconjugate). Conjugates of the antibody and cytotoxic agent are madeusing a variety of bifunctional protein-coupling agents such asN-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP), iminothiolane(IT), bifunctional derivatives of imidoesters (such as dimethyladipimidate HCL), active esters (such as disuccinimidyl suberate),aldehydes (such as glutareldehyde), bis-azido compounds (such as bis(p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such asbis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such astolyene 2,6-diisocyanate), and bis-active fluorine compounds (such as1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin canbe prepared as described in Vitetta et al., Science, 238: 1098 (1987).Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent forconjugation of radionucleotide to the antibody. See WO94/11026.

In another embodiment, the antibody can be conjugated to a “receptor”for utilization in tumor pretargeting wherein the antibody-receptorconjugate is administered to the patient, followed by removal of unboundconjugate from the circulation using a clearing agent and thenadministration of a “ligand” that is in turn conjugated to a cytotoxicagent.

Immunoliposomes

The antibodies disclosed herein can also be formulated asimmunoliposomes. Liposomes containing the antibody are prepared bymethods known in the art, such as described in Epstein et al., Proc.Natl. Acad. Sci. USA, 82: 3688 (1985); Hwang et al., Proc. Natl Acad.Sci. USA, 77: 4030 (1980); and U.S. Pat. Nos. 4,485,045 and 4,544,545.Liposomes with enhanced circulation time are disclosed in U.S. Pat. No.5,013,556.

Particularly useful liposomes can be generated by the reverse-phaseevaporation method with a lipid composition comprisingphosphatidylcholine, cholesterol, and PEG-derivatizedphosphatidylethanolamine (PEG-PE). Liposomes are extruded throughfilters of Chem. 257: 286-288 (1982) via a disulfide-interchangereaction. A chemotherapeutic agent (such as Doxorubicin) is optionallycontained within the liposome. See Gabizon et al., J. National CancerInst., 81(19): 1484 (1989).

A therapeutically effective amount of an antibody of the inventionrelates generally to the amount needed to achieve a therapeuticobjective. As noted above, this may be a binding interaction between theantibody and its target antigen that, in certain cases, interferes withthe functioning of the target, and in other cases, promotes aphysiological response. The amount required to be administered willfurthermore depend on the binding affinity of the antibody for itsspecific antigen, and will also depend on the rate at which anadministered antibody is depleted from the free volume other subject towhich it is administered. Common ranges for therapeutically effectivedosing of an antibody or antibody fragment of the invention may be, byway of nonlimiting example, from about 0.1 mg/kg body weight to about 50mg/kg body weight. Common dosing frequencies may range, for example,from twice daily to once a week.

Antibodies specifically binding a protein of the invention, as well asother molecules identified by the screening assays disclosed herein, canbe administered for the treatment of various disorders in the form ofpharmaceutical compositions. Principles and considerations involved inpreparing such compositions, as well as guidance in the choice ofcomponents are provided, for example, in Remington: The Science AndPractice Of Pharmacy 19th ed. (Alfonso R. Germain, et al., editors) MackPub. Co., Easton, Pa. 1995; Drug Absorption Enhancement: Concepts,Possibilities, Limitations, And Trends, Harwood Academic Publishers,Langhorne, Pa., 1994; and Peptide And Protein Drug Delivery (Advances InParenteral Sciences, Vol. 4), 1991, M. Dekker, New York.

The active ingredients can also be entrapped in microcapsules prepared,for example, by coacervation techniques or by interfacialpolymerization, for example, hydroxymethylcellulose orgelatin-microcapsules and poly-(methylmethacrylate) microcapsules,respectively, in colloidal drug delivery systems (for example,liposomes, albumin microspheres, microemulsions, nano-particles, andnanocapsules) or in macroemulsions. The formulations to be used for invivo administration must be sterile. This is readily accomplished byfiltration through sterile filtration membranes.

Sustained-release preparations can be prepared. Suitable examples ofsustained-release preparations include semipermeable matrices of solidhydrophobic polymers containing the antibody, which matrices are in theform of shaped articles, e.g., films, or microcapsules. Examples ofsustained-release matrices include polyesters, hydrogels (for example,poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides(U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid andgamma-ethyl-L-glutamate, non-degradable ethylene-vinyl acetate,degradable lactic acid-glycolic acid copolymers such as the LUPRONDEPOT™ (injectable microspheres composed of lactic acid-glycolic acidcopolymer and leuprolide acetate), and poly-D-(−)-3-hydroxybutyric acid.While polymers such as ethylene-vinyl acetate and lactic acid-glycolicacid enable release of molecules for over 100 days, certain hydrogelsrelease proteins for shorter time periods.

ELISA Assay

An agent for detecting an analyte protein is an antibody capable ofbinding to an analyte protein, preferably an antibody with a detectablelabel. Antibodies can be polyclonal, or more preferably, monoclonal. Anintact antibody, or a fragment thereof (e.g., Fab or F(ab)2) can beused. The term “labeled”, with regard to the probe or antibody, isintended to encompass direct labeling of the probe or antibody bycoupling (i e., physically linking) a detectable substance to the probeor antibody, as well as indirect labeling of the probe or antibody byreactivity with another reagent that is directly labeled. Examples ofindirect labeling include detection of a primary antibody using afluorescently-labeled secondary antibody and end-labeling of a DNA probewith biotin such that it can be detected with fluorescently-labeledstreptavidin. The term “biological sample” is intended to includetissues, cells and biological fluids isolated from a subject, as well astissues, cells and fluids present within a subject. Included within theusage of the term “biological sample”, therefore, is blood and afraction or component of blood including blood serum, blood plasma, orlymph. That is, the detection method of the invention can be used todetect an analyte mRNA, protein, or genomic DNA in a biological samplein vitro as well as in vivo. For example, in vitro techniques fordetection of an analyte mRNA include Northern hybridizations and in situhybridizations. In vitro techniques for detection of an analyte proteininclude enzyme linked immunosorbent assays (ELISAs), Western blots,immunoprecipitations, and immunofluorescence. In vitro techniques fordetection of an analyte genomic DNA include Southern hybridizations.Procedures for conducting immunoassays are described, for example in“ELISA: Theory and Practice: Methods in Molecular Biology”, Vol. 42, J.R. Crowther (Ed.) Human Press, Totowa, N.J., 1995; “Immunoassay”, E.Diamandis and T. Christopoulus, Academic Press, Inc., San Diego, Calif.,1996; and “Practice and Theory of Enzyme Immunoassays”, P. Tijssen,Elsevier Science Publishers, Amsterdam, 1985. Furthermore, in vivotechniques for detection of an analyte protein include introducing intoa subject a labeled anti-an analyte protein antibody. For example, theantibody can be labeled with a radioactive marker whose presence andlocation in a subject can be detected by standard imaging techniquesintracavity, or transdermally, alone or with effector cells.

Therapeutic Uses and Formulations

The nucleic acids and proteins of the invention are useful in potentialprophylactic and therapeutic applications implicated in a variety ofdisorders including, but not limited to: metabolic disorders, diabetes,obesity, infectious disease, anorexia, cancer, neurodegenerativedisorders, Alzheimer's Disease, Parkinson's Disorder, immune disorders,hematopoictic disorders, and the various dyslipidemias, metabolicdisturbances associated with obesity, the metabolic syndrome X andwasting disorders associated with chronic diseases and various cancers,cardiomyopathy, atherosclerosis, hypertension, congenital heart defects,aortic stenosis, atrial septal defect (ASD), atrioventricular (A-V)canal defect, ductus arteriosus, pulmonary stenosis, subaortic stenosis,ventricular septal defect (VSD), valve diseases, tuberous sclerosis,scleroderma, lupus erythematosus, obesity, transplantation,adrenoleukodystrophy, congenital adrenal hyperplasia, prostate cancer,neoplasm; adenocarcinoma, lymphoma, uterus cancer, fertility, leukemia,hemophilia, hypercoagulation, idiopathic thrombocytopenic purpura,immunodeficiencies, graft versus host disease, AIDS, bronchial asthma,rheumatoid and osteoarthritis, Crohn's disease; multiple sclerosis,treatment of Albright Hereditary Ostoeodystrophy, and other diseases,disorders and conditions of the like.

Preparations for administration of the therapeutic complex of theinvention include sterile aqueous or non-aqueous solutions, suspensions,and emulsions. Examples of non-aqueous solvents are propylene glycol,polyethylene glycol, vegetable oils such as olive oil, and injectableorganic esters such as ethyl oleate. Aqueous carriers include water,alcoholic/aqueous solutions, emulsions or suspensions, including salineand buffered media. Vehicles include sodium chloride solution, Ringer'sdextrose, dextrose and sodium chloride, lactated Ringer's intravenousvehicles including fluid and nutrient replenishers, electrolytereplenishers, and the like. Preservatives and other additives may beadded such as, for example, antimicrobial agents, anti-oxidants,chelating agents and inert gases and the like.

The nucleic acid molecules, polypeptides, and antibodies (also referredto herein as “active compounds”) of the invention, and derivatives,fragments, analogs and homologs thereof, can be incorporated intopharmaceutical compositions suitable for administration. Suchcompositions typically comprise the nucleic acid molecule, protein, orantibody and a pharmaceutically acceptable carrier. As used herein,“pharmaceutically acceptable carrier” is intended to include any and allsolvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents, and the like,compatible with pharmaceutical administration. Suitable carriers aredescribed in the most recent edition of Remington's PharmaceuticalSciences, a standard reference text in the field, which is incorporatedherein by reference. Preferred examples of such carriers or diluentsinclude, but are not limited to, water, saline, finger's solutions,dextrose solution, and 5% human serum albumin. Liposomes and non-aqueousvehicles such as fixed oils may also be used. The use of such media andagents for pharmaceutically active substances is well known in the art.Except insofar as any conventional media or agent is incompatible withthe active compound, use thereof in the compositions is contemplated.Supplementary active compounds can also be incorporated into thecompositions.

A pharmaceutical composition of the invention is formulated to becompatible with its intended route of administration. Examples of routesof administration include parenteral, e.g., intravenous, intradermal,subcutaneous, oral (e.g., inhalation), transdermal (i.e., topical),transmucosal, intraperitoneal, and rectal administration. Solutions orsuspensions used for parenteral, intradermal, or subcutaneousapplication can include the following components: a sterile diluent suchas water for injection, saline solution, fixed oils, polyethyleneglycols, glycerine, propylene glycol or other synthetic solvents;antibacterial agents such as benzyl alcohol or methyl parabens;antioxidants such as ascorbic acid or sodium bisulfite; chelating agentssuch as ethylenediaminetetraacetic acid (EDTA); buffers such asacetates, citrates or phosphates, and agents for the adjustment oftonicity such as sodium chloride or dextrose. The pH can be adjustedwith acids or bases, such as hydrochloric acid or sodium hydroxide. Theparenteral preparation can be enclosed in ampoules, disposable syringesor multiple dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersion. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, Cremophor™.(BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In allcases, the composition must be sterile and should be fluid to the extentthat easy syringeability exists. It must be stable under the conditionsof manufacture and storage and must be preserved against thecontaminating action of microorganisms such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, andliquid polyethylene glycol, and the like), and suitable mixtures thereofThe proper fluidity can be maintained, for example, by the use of acoating such as lecithin, by the maintenance of the required particlesize in the case of dispersion and by the use of surfactants. Preventionof the action of microorganisms can be achieved by various antibacterialand antifungal agents, for example, parabens, chlorobutanol, phenol,ascorbic acid, thimerosal, and the like. In many cases, it will bepreferable to include isotonic agents, for example, sugars, polyalcoholssuch as manitol, sorbitol, sodium chloride in the composition. Prolongedabsorption of the injectable compositions can be brought about byincluding in the composition an agent which delays absorption, forexample, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the activecompound (e.g., the therapeutic complex of the invention) in therequired amount in an appropriate solvent with one or a combination ofingredients enumerated above, as required, followed by filteredsterilization. Generally, dispersions are prepared by incorporating theactive compound into a sterile vehicle that contains a basic dispersionmedium and the required other ingredients from those enumerated above.In the case of sterile powders for the preparation of sterile injectablesolutions, methods of preparation are vacuum drying and freeze-dryingthat yields a powder of the active ingredient plus any additionaldesired ingredient from a previously sterile-filtered solution thereof.

Oral compositions generally include an inert diluent or an ediblecarrier. They can be enclosed in gelatin capsules or compressed intotablets. For the purpose of oral therapeutic administration, the activecompound can be incorporated with excipients and used in the form oftablets, troches, or capsules. Oral compositions can also be preparedusing a fluid carrier for use as a mouthwash, wherein the compound inthe fluid carrier is applied orally and swished and expectorated orswallowed. Pharmaceutically compatible binding agents, and/or adjuvantmaterials can be included as part of the composition. The tablets,pills, capsules, troches and the like can contain any of the followingingredients, or compounds of a similar nature: a binder such asmicrocrystalline cellulose, gum tragacanth or gelatin; an excipient suchas starch or lactose, a disintegrating agent such as alginic acid,Primogel, or corn starch; a lubricant such as magnesium stearate orSterotes; a glidant such as colloidal silicon dioxide; a sweeteningagent such as sucrose or saccharin; or a flavoring agent such aspeppermint, methyl salicylate, or orange flavoring.

For oral administration, the pharmaceutical compositions may take theform for example, tablets or capsules prepared by conventional meanswith pharmaceutically acceptable excipients such as binding agents(e.g., pregelatinised maize starch, polyvinylpyrrolidone orhydroxypropyl methylcellulose); fillers (e.g., lactose, microcrystallinecellulose or calcium hydrogen phosphate); lubricants (e.g., magnesiumstearate, talc or silica); disintegrants (e.g., potato starch or sodiumstarch glycolate); or wetting agents (e.g., sodium lauryl sulphate). Thetablets may be coated by methods well known in the art. Liquidpreparations for oral administration may take the form of, for example,solutions, syrups, or suspensions, or they may be presented as a dryproduct for constitution with water or other suitable vehicle beforeuse. Such liquid preparations may be prepared by conventional means withpharmaceutically acceptable additives such as suspending agents (e.g.,sorbitol syrup, cellulose derivatives or hydrogenated edible fats);emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles(e.g., almond oil, oily esters, ethyl alcohol or fractionated vegetableoils); and preservatives (e.g., methyl or propyl-p-hydroxybenzoates orsorbic acid). The preparations may also contain buffer salts, flavoring,coloring, and sweetening agents as appropriate.

Preparations for oral administration may be suitably formulated to givecontrolled release of the active compound. For buccal administration thecompositions may take the form of tablets or lozenges formulated inconventional manner. For administration by inhalation, the compounds foruse according to the present invention are conveniently delivered in theform of an aerosol spray presentation from pressurized packs or anebuliser, with the use of a suitable propellant, e.g.,dichlorodifluoromethane, trichlorofluoromethane,dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In thecase of a pressurized aerosol the dosage unit may be determined byproviding a valve to deliver a metered amount. Capsules and cartridgesof e.g. gelatin for use in an inhaler or insufflator may be formulatedcontaining a powder mix of the compound and a suitable powder base suchas lactose or starch. The compounds may be formulated for parenteraladministration by injection, e.g., by bolus injection or continuousinfusion. Formulations for injection may be presented in unit dosageform, e.g., in ampoules or in multi-dose containers, with an addedpreservative. The compositions may take such forms as suspensions,solutions, or emulsions in oily or aqueous vehicles, and may containformulatory agents such as suspending, stabilizing, and/or dispersingagents. Alternatively, the active ingredient may be in powder form forconstitution with a suitable vehicle, e.g., sterile pyrogen-free water,before use. The compounds may also be formulated in rectal compositionssuch as suppositories or retention enemas, e.g., containing conventionalsuppository bases such as cocoa butter or other glycerides. In additionto the formulations described previously, the compounds may also beformulated as a depot preparation. Such long acting formulations may beadministered by implantation (for example subcutaneously orintramuscularly) or by intramuscular injection. Thus, for example, thecompounds may be formulated with suitable polymeric or hydrophobicmaterials (for example as an emulsion in an acceptable oil) or ionexchange resins, or as sparingly soluble derivatives, for example, as asparingly soluble salt.

For administration by inhalation, the compounds are delivered in theform of an aerosol spray from pressured container or dispenser whichcontains a suitable propellant, e.g., a gas such as carbon dioxide, or anebulizer.

Systemic administration can also be by transmucosal or transdermalmeans. For transmucosal or transdermal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art, and include, forexample, for transmucosal administration, detergents, bile salts, andfusidic acid derivatives. Transmucosal administration can beaccomplished through the use of nasal sprays or suppositories. Fortransdermal administration, the active compounds are formulated intoointments, salves, gels, or creams as generally known in the art.

In one embodiment, the active compounds are prepared with carriers thatwill protect the compound against rapid elimination from the body, suchas a controlled release formulation, including implants andmicroencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid.Methods for preparation of such formulations will be apparent to thoseskilled in the art. The materials can also be obtained commercially fromAlza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions(including liposomes targeted to infected cells with monoclonalantibodies to viral antigens) can also be used as pharmaceuticallyacceptable carriers. These can be prepared according to methods known tothose skilled in the art, for example, as described in U.S. Pat. No.4,522,811.

It is especially advantageous to formulate oral or parenteralcompositions in dosage unit form for ease of administration anduniformity of dosage. Dosage unit form as used herein refers tophysically discrete units suited as unitary dosages for the subject tobe treated; each unit containing a predetermined quantity of activecompound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms of the invention are dictated by and directlydependent on the unique characteristics of the active compound and theparticular therapeutic effect to be achieved, and the limitationsinherent in the art of compounding such an active compound for thetreatment of individuals.

The nucleic acid molecules of the invention can be inserted into vectorsand used as gene therapy vectors. Gene therapy vectors can be deliveredto a subject by, for example, intravenous injection, localadministration (see, e.g., U.S. Pat. No. 5,328,470) or by stereotacticinjection (see, e.g., Chen, et al., 1994. Proc. Natl. Acad. Sci. USA 91:3054-3057). The pharmaceutical preparation of the gene therapy vectorcan include the gene therapy vector in an acceptable diluent, or cancomprise a slow release matrix in which the gene delivery vehicle isimbedded. Alternatively, where the complete gene delivery vector can beproduced intact from recombinant cells, e.g., retroviral vectors, thepharmaceutical preparation can include one or more cells that producethe gene delivery system. The pharmaceutical compositions can beincluded in a container, pack, or dispenser together with instructionsfor administration.

A therapeutically effective dose refers to that amount of thetherapeutic complex sufficient to result in amelioration or delay ofsymptoms. Toxicity and therapeutic efficacy of such compounds can bedetermined by standard pharmaceutical procedures in cell cultures orexperimental animals, e.g., for determining the LD50 (the dose lethal to50% of the population) and the ED50 (the dose therapeutically effectivein 50% of the population). The dose ratio between toxic and therapeuticeffects is the therapeutic index and it can be expressed as the ratioLD50/ED50. Compounds that exhibit large therapeutic indices arepreferred. While compounds that exhibit toxic side effects may be used,care should be taken to design a delivery system that targets suchcompounds to the site of affected tissue in order to minimize potentialdamage to uninfected cells and, thereby, reduce side effects. The dataobtained from the cell culture assays and animal studies can be used informulating a range of dosage for use in humans. The dosage of suchcompounds lies preferably within a range of circulating concentrationsthat include the ED50 with little or no toxicity. The dosage may varywithin this range depending upon the dosage form employed and the routeof administration utilized. For any compound used in the method of theinvention, the therapeutically effective dose can be estimated initiallyfrom cell culture assays. A dose may be formulated in animal models toachieve a circulating plasma concentration range that includes the IC50(i.e., the concentration of the test compound which achieves ahalf-maximal inhibition of symptoms) as determined in cell culture. Suchinformation can be used to more accurately determine useful doses inhumans. Levels in plasma may be measured, for example, by highperformance liquid chromatography.

Pharmaceutical compositions may be formulated in conventional mannerusing one or more physiologically acceptable carriers or excipients.Thus, the compounds and their physiologically acceptable salts andsolvates may be formulated for administration by inhalation orinsufflation (either through the mouth or the nose) or oral, buccal,intravenous, intraperitoneal, parenteral or rectal administration.

Also disclosed according to the present invention is a kit or systemutilizing any one of the methods, selection strategies, materials, orcomponents described herein. Exemplary kits according to the presentdisclosure will optionally, additionally include instructions forperforming methods or assays, packaging materials, one or morecontainers which contain an assay, a device or system components, or thelike.

In an additional aspect, the present invention provides kits embodyingthe complex and methods of using disclosed herein. Kits of the inventionoptionally include one or more of the following: (1) polypeptide ornucleic acid components described herein; (2) instructions forpracticing the methods described herein, and/or for operating theselection procedure herein; (3) one or more detection assay components;(4) a container for holding nucleic acids or polypeptides, other nucleicacids, transgenic plants, animals, cells, or the like and, (5) packagingmaterials.

Transgenic Organisms

A transgenic cell or animal used in the methods of the invention caninclude a transgene that encodes, e.g., a copy of a chimeric polypeptidecomprising an interleukin and interleukin receptor. The transgene canencode a protein that is normally exogenous to the transgenic cell oranimal, including a human protein. The transgene can be linked to aheterologous or a native promoter.

This disclosure further relates to a method of producing transgenicanimals. Techniques known in the art may be used to introduce thetransgene into animals to produce the founder line of animals. Suchtechniques include, but are not limited to: pronuclear microinjection;retrovirus mediated gene transfer into germ lines (Van der Putten etal., Proc. Natl. Acad. Sci. USA 82: 6148-6152, 1985; gene targeting inembryonic stem cells (Thompson et al., Cell 56: 313-321, 1989;electroporation of embryos (Lo, Mol. Cell Biol. 3: 1803-1814, 1983; andsperm-mediated gene transfer (Lavitrano, et al., Cell 57: 717-723, 1989;etc. For a review of such techniques, see Gordon, Intl. Rev. Cytol. 115:171-229, 1989. Accordingly, the invention features a transgenic organismthat contains a transgene encoding a chimeric interleukin/interleukinreceptor polypeptide. The transgenic organism can be a eukaryotic cell,for example, a yeast cell, an insect, e.g., a worm or a fly, a fish, areptile, a bird, or a mammal, e.g., a rodent. The transgenic organismcan further comprise a genetic alteration, e.g., a point mutation,insertion, or deficiency, in an endogenous gene.

A host cell of the invention, such as a prokaryotic or eukaryotic hostcell in culture, can be used to produce (i.e., express) the polypeptidecomponents or complex of the invention. Accordingly, the inventionfurther provides methods for producing protein using the host cells ofthe invention. In one embodiment, the method comprises culturing thehost cell of invention (into which a recombinant expression vectorencoding the protein has been introduced) in a suitable medium such thatthe protein is produced. In another embodiment, the method furthercomprises isolating the protein from the medium or the host cell.

Another aspect of the invention pertains to host cells into which arecombinant expression vector of the invention has been introduced. Theterms “host cell” and “recombinant host cell” are used interchangeablyherein. It is understood that such terms refer not only to theparticular subject cell but also to the progeny or potential progeny ofsuch a cell. Because certain modifications may occur in succeedinggenerations due to either mutation or environmental influences, suchprogeny may not, in fact, be identical to the parent cell, but are stillincluded within the scope of the term as used herein. A host cell can beany prokaryotic or eukaryotic cell. For example, protein can beexpressed in bacterial cells such as E. coli, insect cells, yeast ormammalian cells (such as Chinese hamster ovary cells (CHO) or COScells). Other suitable host cells are known to those skilled in the art.

Vector DNA can be introduced into prokaryotic or eukaryotic cells viaconventional transformation or transfection techniques. As used herein,the terms “transformation” and “transfection” are intended to refer to avariety of art-recognized techniques for introducing foreign nucleicacid (e.g., DNA) into a host cell, including calcium phosphate orcalcium chloride co-precipitation, DEAE-dextran-mediated transfection,lipofection, or electroporation. Suitable methods for transforming ortransfecting host cells can be found in Sambrook, et al. (MOLECULARCLONING: A LABORATORY MANUAL. 2nd ed., Cold Spring Harbor Laboratory,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989),and other laboratory manuals.

For stable transfection of mammalian cells, it is known that, dependingupon the expression vector and transfection technique used, only a smallfraction of cells may integrate the foreign DNA into their genome. Inorder to identify and select these integrants, a gene that encodes aselectable marker (e.g., resistance to antibiotics) is generallyintroduced into the host cells along with the gene of interest. Variousselectable markers include those that confer resistance to drugs, suchas G418, hygromycin and methotrexate. Nucleic acid encoding a selectablemarker can be introduced into a host cell on the same vector as thatencoding the protein or can be introduced on a separate vector. Cellsstably transfected with the introduced nucleic acid can be identified bydrug selection (e.g., cells that have incorporated the selectable markergene will survive, while the other cells die).

The host cells of the invention can also be used to produce non-humantransgenic animals. For example, in one embodiment, a host cell of theinvention is a fertilized oocyte or an embryonic stem cell into whichthe protein-coding sequences have been introduced. Such host cells canthen be used to create non-human transgenic animals in which exogenouspolypeptide sequences have been introduced into their genome orhomologous recombinant animals in which endogenous polypeptide sequenceshave been altered. Such animals are useful for studying the functionand/or activity of proteins and for identifying and/or evaluatingmodulators of protein activity. As used herein, a “transgenic animal” isa non-human animal, preferably a mammal, more preferably a rodent suchas a rat or mouse, in which one or more of the cells of the animalincludes a transgene. Other examples of transgenic animals includenon-human primates, sheep, dogs, cows, goats, chickens, amphibians, etc.A transgene is exogenous DNA that is integrated into the genome of acell from which a transgenic animal develops and that remains in thegenome of the mature animal, thereby directing the expression of anencoded gene product in one or more cell types or tissues of thetransgenic animal. As used herein, a “homologous recombinant animal” isa non-human animal, preferably a mammal, more preferably a mouse, inwhich an endogenous gene has been altered by homologous recombinationbetween the endogenous gene and an exogenous DNA molecule introducedinto a cell of the animal, e.g., an embryonic cell of the animal, priorto development of the animal.

An example of a preferred embodiment of the invention is provided below.As will be understood by one of ordinary skill in the art, thetechniques described and hereby incorporated into the present inventionare generally applicable and may be varied in any number of ways withoutdeparting from the general scope of the invention. The following exampleis given by way of example of the preferred embodiments, and is in noway considered to be limiting to the invention. For example, therelative quantities of the ingredients may be varied to achievedifferent desired effects, additional ingredients may be added, and/orsimilar ingredients may be substituted for one or more of theingredients described.

EXAMPLE 1

Co-Administration of IL-15 and IL-15Ra Drives CD8 Memory T Cell and NKCell Proliferation In Vivo

In order to determine whether co-administration of IL-15 and recombinantmouse IL-15Ra-Fc (rmIL-15Ra-Fc) could mediate IL-15 activity in vivo, weutilized an adoptive transfer model to gage the effect of IL-15 on theproliferation of CD8+ T cells. CD45.1 CFSE labeled enriched splenic CD8+T cells were transferred to normal CD45.2 mice and rmIL-15Ra-Fc (about15 μg). Four days after treatment with IL-15 alone, about 11% of thedonor CD8+ T cell population had divided (FIG. 1 a, top panels), inagreement with our previous results. In dramatic contrast, thecoadministration of the same amount of IL-15 bound to rmIL-15Ra-Fcresulted in the proliferation of about 69% of the donor CD8+ T cells(FIG. 1). Furthermore, while the majority of CD8 T cells responding toIL-15 alone divided once, the cells responding to combination treatmentunderwent 5-7 divisions, resulting in a substantial increase in cellnumbers (data not shown). The bulk of the dividing cells expressed highlevels of CD44, suggesting that the responding cells were primarilymemory CD8+ T cells or that CD44 had been upregulated (FIG. 1 a, bottompanels). Importantly, administration of rmIL-15Ra-Fc alone did notinduce proliferation of CD8+ T cells (data not shown). Of note,co-administration of a soluble form of rmIL-15Ra with IL-15 alsoresulted in enhanced proliferation of donor CD8+ T cells albeit to alevel intermediate to IL-15 alone and IL-15 combined with rmIL-15Ra-Fc(data not shown). In order to test the action of combined therapy onbona-fide memory CD8 T cells, we adoptively transferred CFSE-labeledovalbumin (OVA)-specific CD8+ memory T cells that had been generated byinfection with recombinant vesicular stomatitis virus expressing OVA(VSV-OVA). Similar to the above results, antigen-specific memory CD8+ Tcells responding to combined IL-15/IL-15Ra-Fc treatment proliferated toa much greater extent than those provided IL-15 alone (FIG. 1b ).

Past studies have implicated IL-15 as an inducer of B cell, NK cell andNK T cell proliferation, but not of CD4+ T cell proliferation.Therefore, we examined the ability of IL-15 and receptor-complexed IL-15to induce proliferation of these cell types using the adoptive transfersystem. CD4+ T cells and B cells did not proliferate in response toabout 2.5 μg of IL-15, while NK cells proliferated very little (FIG. 2).In contrast, coadministration of rmIL-15Ra-Fc with IL-15 inducedextensive proliferation of NK cells while B cells did not respond. Theresponse of NK-T cells was similar to that of NK cells (data not shown).Interestingly, although IL-15 is not thought to mediate proliferation ofmouse CD4+ T cells, CD4+ T cells illustrated an intermediate response tothe administered complex.

EXAMPLE 2

Complexed IL-15/IL-15Ra Greatly Enhances IL-15 Activity In Vivo

We next examined the early kinetics of the proliferative response to thecoadministration of rmIL-15Ra-Fc with IL-15. CFSE dilution wasnegligible one day after treatment, but by day two about 36% of thedonor CD8+ T cell population had divided, with an appreciable number ofcells in the third and fourth rounds of division (FIG. 3). By day threeabout 59% of donor CD8+ T cells had divided with many cells in divisions5-6, while about 73% had divided by day four with some cells in theseventh round of division. These results and others showed that themaximum effect of a single dose of IL-15/rmIL-15Ra-Fc was achieved byapproximately 4 days post-treatment, followed by the donor CD8+ T cellsentering a protracted rate of proliferation characteristic of memoryCD8+ T cells (data not shown).

In order to obtain an approximation of the enhancement of activityobtained by combined treatment over that of IL-15 alone we performedtitrations of IL-15 and IL-15/rmIL-15Ra-Fc using the adoptive transfermodel. Comparisons were based on the extent of donor CD8+ T cellproliferation as assessed by CFSE dilution. A dose of about 0.1 μg ofIL-15 combined with about 0.6 μg of IL-15Ra-Fc induced a level ofproliferation similar to that of about 5 μg of IL-15 (FIG. 4a ). Thus,in this type of experiment, IL-15 activity was enhanced ˜50-fold bycoadministration with rmIL-15Ra-Fc. Considering this substantialenhancement, we questioned whether IL-15 alone could achieve this levelof activity. Even with the administration of about 37.5 μg of IL-15, thelevel of proliferation obtained with about 0.5 μg of receptor complexedIL-15 could not be achieved (FIG. 4b ). These results suggested thatIL-15Ra availability may be limiting in vivo since increasing IL-15levels did not result in further augmentation of activity.

EXAMPLE 3

Complexed IL-15/IL-15Ra Operates Via Transpresentation Requiring IL-15Rb

The effects of complexed IL-15/IL-15Ra could either be mediated bydirect or indirect effects on the responding cell types. If direct, thenit might be expected that the target cells would be required to expressIL-15R component(s). To test this, we transferred CFSE-labeledIL-15Ra−/− CD8+ T cells into IL-15Ra−/− hosts and treated the mice witheither IL-15 or complexed IL-15/IL-15Ra. IL-15 could not betranspresented in the absence of IL-15Ra, and did not induceproliferation (FIG. 5a ). On the other hand, donor CD8+ T cells fromIL-15/rmIL-15Ra-Fc treated mice proliferated extensively. Furthermore,the IL-15Ra−/− donor cells, which primarily consisted of nave phenotypeCD8+ T cells, progressively increased their expression of CD44 and CD122with division. Since responding T cells did not require IL-15Ra torespond to complexed IL-15/IL-15Ra, we examined the role of IL-15Rb(CD122) in mediating this effect. To this end, we transferredCFSE-labeled CD122+/+ or CD122−/− CD8+ T cells into normal mice andanalyzed the donor cells for CFSE dilution 4 days after treatment. Whilecontrol cells proliferated vigorously in response to IL-15/rmIL-15Ra-Fctreatment, CD122−/− donor CD8+ T cells did not proliferate in responseto coadministration (FIG. 5b ). Taken together, the results indicatedthat IL-15/IL-15Ra-Fc operated via direct transpresentation throughinteraction with the IL-15Rb likely in conjunction with gammaC.

EXAMPLE 4

Proliferation Induced by Forced IL-15 Transpresentation Requires MHCclass I But Not IL-7 or Dendritic Cells

While naïve T cells require MHC class I and endogenous peptide for theirsurvival, memory CD8 T cell survival and proliferation is thought to beMHC class I-independent. Homeostatic proliferation of these subsets inempty hosts exhibits similar MHC requirements. Given these results, itwas important to determine the MHC requirement for proliferation inducedby co-administration of rmIL-15Ra-Fc with IL-15. Thus, we cotransferrednaïve TCR transgenic CD8+ T cells (OT-I) and enriched B6 CD8+ T cells(which contain memory cells) to normal or MHC class I deficient(b2-microglobulin−/−) mice. Interestingly, naïve OT-IRAG−/− CD8 T cellsproliferated robustly in response to treatment with the complex in a MHCclass I sufficient host. In contrast, in MHC class I−/− hosts, naïve Tcell proliferation did not occur (FIG. 6a ). Similarly, B6 CD8 T cellsproliferated in normal hosts but surprisingly proliferation wasvirtually absent in MHC class I−/− hosts. These data indicated thatinduction of proliferation by IL-15/IL-15Ra was MHC class I dependentfor both naïve and memory CD8+ T cells. Since the proliferative responseinduced by the coadministration of rmIL-15Ra-Fc with IL-15 was dependenton host expression of MHC class I we wished to investigate othercriteria that might also play a role. We examined the involvement ofIL-7, since this cytokine is essential for homeostatic proliferation ofCD8 T cells in immunodeficient hosts. CFSE-labeled CD8+ T cells weretransferred to control or IL-7−/− mice and combined IL-15/rmIL-15Ra-Fcwas administered. In the presence or absence of IL-7 CD8+ T cellsproliferated equally well in response to IL-15 with IL-15Ra-Fc (FIG. 6b), indicating that IL-7 was not involved in IL-15 mediated proliferationin our system. Previous studies have highlighted the potential ofdendritic cells (DC) in mediating IL-15 activity and MHC expression byDC can be important in T cell homeostasis. To test what role DC play inthe proliferative response induced by IL-15/IL-15Ra coadministration weutilized a system in which DC can be conditionally depleted. CD11c-DTRmice express the simian diptheria toxin receptor under the control ofthe CD11c promoter, making CD11c+ cells susceptible to DT, whichremoves >95% of DC. Due to the toxicity of DT to intact CD11c-DTR miceas a result of effects on non-hematopoietic cells, we generated chimerasusing CD11c-DTR bone marrow and normal B6 hosts. CFSE-labeled CD8+ Tcells were then transferred to the chimeras which were treated with DTprior to administration of the IL-15/IL-15Ra complex. Interestingly, wefound no difference in CD8+ T cell proliferation between DT treatedcontrol or CD11c-DTR chimeras (FIG. 6c ). Thus, although MHC class I wasessential for IL-15 mediated proliferation, DC were not required.

EXAMPLE 5

IL-15/IL-15Ra Immunotherapy Induces Naïve T Cell Activation and EffectorFunction

In previous experiments we noted that CD44 low polyclonal CD8 T cells aswell as naïve TCR transgenic T cells responded to IL-15 whenco-administered with IL-15Ra-Fc (FIGS. 5 and 6). Considering that underhomeostatic conditions, CD8 memory T cells exhibit much greaterresponsiveness to IL-15 than do naïve CD8+ T cells, we wished todirectly compare the responsiveness of these two subsets to complexedIL-15/rmIL-15Ra-Fc. To do so, CFSE-labeled memory OT-I and naïve OT-ICD8+ T cells were adoptively transferred into the same congenic C57BL/6hosts and proliferation was analyzed 4 days after treatment withIL-15/IL-15Ra-Fc. Surprisingly, naïve OT-I CD8 T cells proliferatedalmost as well as memory OT-I CD8+ T cells (FIG. 7a ). The naïve OT-Icells also expanded ˜10-fold in response to the complex as compared tocontrols and upregulated CD44 (FIG. 7b ). In light of the robustproliferation induced in naïve T cells, it was of interest to establishwhether effector function was concomitantly induced. To test thisquestion we adoptively transferred naïve OT-I CD8+ T cells into congenicC57BL/6 hosts and, using an in vivo killing assay, measured antigenspecific lytic activity four days after treatment withIL-15/rmIL-15Ra-Fc or after infection with recombinant vesicularstomatitis virus expressing ovalbumin (VSV-OVA) for comparison.Interestingly, IL-15/rmIL-15Ra-Fc treatment resulted in induction ofrobust antigen-specific lytic activity, similar to the level obtainedwith virus infection (FIG. 7c ). In addition to lytic activity, themajority of naïve OT-I CD8+ T cells activated by IL-15/IL-15Ra-Fc orVSV-OVA infection produced high levels of IFNg, following in vitrorestimulation with peptide (FIG. 7d ). This result was in contrast tothe negligible frequency of OT-I cells producing IFNg from control (PBS)and IL-15 treated mice (FIG. 7d ). Thus, the induction of effectorfunction in naïve CD8+ T cells by co-administration of IL-15Ra-Fc withIL-15 paralleled the activation obtained by infection.

EXAMPLE 6

Treatment of Naïve T Cells With Complexed IL-15/IL-15Ra-Fc GeneratesMemory CD8+ T Cells

Although naïve T cells developed into effector cells in response totranspresented IL-15, it remained to be seen whether this was atransient effect or resulted in memory T cell development. Therefore, weanalyzed the number and phenotype of OT-I T cells 44 days after naïveOT-I T cell transfer and IL-15/IL-15Ra-Fc treatment. At this time pointa ˜5-fold higher percentage of OT-I cells was present followingIL-15/IL-15Ra administration as compared to untreated mice (FIG. 8, toppanels). Moreover, nearly all of these cells expressed high levels ofCD44 and CD122 (FIG. 8, middle and bottom panels). Thus, even in theabsence of antigen, IL-15/IL-15Ra-Fc treatment was able to induce thedevelopment of memory CD8+ T cells.

Recent findings support the use of IL-15 as an adjuvant for vaccination,tumor immunotherapy, and immune system reconstitution inimmunodeficiency. In the case of cancer treatment, induction oflymphopenia is now being employed to enhance the functional activity ofadoptively transferred lymphocytes. This modality is based on thefinding that CD8+ T cells undergoing lymphopenia-driven homeostaticproliferation differentiate into effector cells with lytic and cytokineproducing activities. The differentiation of CD8+ T cells to effectorand memory phenotype cells also requires MHC class I expression. Thus,the proliferation and functional activities induced by theIL-15/IL-15Ra-Fc complex in intact hosts mimicked homeostaticproliferation triggered by lymphopenia. Moreover, the level ofproliferation obtained by treatment with the complex could not beachieved by high doses of IL-15 alone. Since the same cell producingIL-15 may also transpresent the cytokine, the availability of freeIL-15Ra may be limited. In addition, the short half-life of IL-15 may beextended when complexed to the receptor. Therefore, treatment with IL-15alone is unlikely to achieve the full therapeutic potential of thecytokine. The combined administration of IL-15/IL-15Ra may circumventthese problems and provide improved efficacy.

The mechanism of action of complexed IL-15/IL-15Ra was of particularinterest given the current paradigm regarding the requirements for naïveand memory T cell homeostatic survival and proliferation. Under normalconditions, survival of both naïve and memory CD8+ T cells requiresIL-7, while IL-15 is essential for homeostatic proliferation of memoryCD8+ T cells and NK cells. In a lymphopenic environment, IL-7 isrequired for homeostatic proliferation of naïve CD8+ and CD4+ T cells,and plays a role, along with IL-15, in mediating CD8+ memory T cellhomeostatic proliferation. Thus, it was unexpected that naïve CD8+ Tcells responded vigorously to the IL-15/IL-15Ra complex. It should benoted however that in IL-15−/− mice, the naïve CD8+ T cell pool isdecreased by about 50%, suggesting that either naïve CD8+ T celldevelopment and/or survival requires IL-15. In any case, proliferationof naïve CD8+ T cells driven by receptor-bound IL-15/IL-15Ra was IL-7independent and required IL-15Ra expression. This result indicated thatnaïve CD8+ T cells expressed sufficient levels of IL-15Ra to respond toIL-15/IL-15Ra but not to soluble IL-15 alone. In addition, naïve CD8+ Tcells acquired effector function and subsequently developed intolong-lived memory CD8+ T cells expressing high levels of CD44 and CD122.Interestingly, IL-15/IL-15Ra triggered activation of naïve or memoryCD8+ T cells required MHC class I expression. While the survival ofnaïve CD8+ T cells is dependent on MHC, the survival of CD8 memory Tcells is believed to be MHC independent. Thus, a requirement for MHCclass I in memory cell proliferation induced by receptor complexed IL-15was somewhat unexpected but supports a role for MHC in aspects of memorycell function, as has been previously demonstrated. Our findingsillustrate the potential power of IL-15 in driving robust NK and CD8+ Tcell expansion and effector differentiation in intact hosts. As with anyadjuvant, it will be necessary to determine whether such activation mayalso enhance autoimmunity. Nevertheless, this system may provide themeans to bolster immune reconstitution in immunodeficiencies or afterbone marrow or stem cell transplantation. Moreover, while adoptiveimmunotherapy in the treatment of cancer may also be augmented byadministration of IL-15/IL-15Ra, it is also possible that treatment withthe complex alone could drive sufficient expansion of endogenousantigen-specific T cells, as well as NK/NKT cells, to provide some levelof protection. Further studies are needed to determine the potential forthis novel complex in immunotherapy.

TABLE 1 Combined IL-15Ra/IL-15 treatment is an effective anti-tumortherapy.^(†) Tumor Treatment Tissue Dose PBS IL-15 IL-15Ra/IL-15 Liver 1× 10⁵ 2-l; 0; 2-l; 3-l ^(‡) 0; 3-l; 3-l; 2-l; 0 0; 0; 0; 0; 0 2 × 10⁵2-l; 4-l; 2-l; 1-m; 2-m 11-m; 0; 1-s; 0; 0; 0; 0; 0 3-l; 5-l 1 × 10⁶24-l; 14; 9-l; 4-l ND 0; 2-s; 2-s; 0 Lung 1 × 10⁵ 0; 0; 0; 2-l ^(‡) 0;1-s; 23-s; 0; 0; 0; 0; 0 4-m; 100-s 2 × 10⁵ 2-m; 2-m; 2-m; 6 (1-l; 5-s);2-m; 3-m; 13 (1-l; 1-s; 3-s; 100-s; 52 (2-l; 50-s) 12-s); 2-m; 50-s 0; 01 × 10⁶ 65-l; 61-l; 81-l; 65-l ND^(£) 28-s; 33-s; 22-s; 42-s Othertumors* 1 × 10⁵ +++ 5/5 +++ 5/5 § 5/5 2 × 10⁵ +++ 3/5; § 2/5 +++ 4/5; §1/5 + 1/5; § 4/5 1 × 10⁶ ++++ 4/4 ND + 1/4; § 3/4 ^(†)The indicated doseof B16-F1 melanoma was given intravenously, and one and 10 days latermice were treated intraperitoneally. with PBS, 2.5 ug IL-15 or 2.5 ugIL-15 + 15 ug sIL-15Ra-Fc. 21 days after tumor inoculation the tumorburden was assessed. Tumor size: (s) ≈ microscopic to 2 mm; (m) ≈ 2-5mm; (l) > 5 mm ^(‡) Died before analysis ^(£)Not done *Includes tumorsin the body cavity including in kidney, pancreas, lymph nodes and othertissues. § = no tumors observed; + = 1 small tumor; +++ = multiplemedium to large tumors; ++++ = large tumor masses.

Exemplary Methods

Mice. C57BL/6-Ly 5.1 mice were purchased from The Jackson Laboratory(Bar Harbor, Me.). C57BL/6-Ly 5.2 mice were purchased from CharlesRiver. The OT-I mouse line was generously provided by Dr. W. R. Heath(WEHI, Parkville, Australia) and Dr. F. Carbone (Monash Medical School,Prahan, Victoria, Australia) and was maintained as a C57BL/6-Ly5.2 lineon a RAG−/− background.

IL-15Ra−/− mice22 were generously provided by Dr. Averil Ma (UCSF).Spleen cells from IL-2Rb−/− mice were generously provided by Dr. MichaelFarrar (UMINN). DTR transgenic mice were a gift from Dr. D. Littman(Skirball Institute, NY, N.Y.). The mice were backcrossed 10 times toC57BL/6 at the UCONN Health Center facilities. DTR Tg+ mice werescreened by PCR of tail DNA as previously described. IL-7−/− mice wereoriginally obtained from DNAX Research Institute of Molecular andCellular Biology (Palo Alto, Calif.) and were maintained on aC57BL/6×129/Ola hybrid background.

IL-15 treatment. Recombinant mouse IL-15Ra-Fc chimeric molecule waspurchased from R&D Systems, Inc. (Minneapolis, Minn.). hIL-15 andrmIL-15Ra-Fc, both suspended in PBS, were mixed and incubated for about30 min at about 37° C. Each mouse, unless specifically noted, received2.5 μg IL-15 and 15 μg rmIL-15Ra-Fc in 200 ul PBS i.p.

Human. A DNA sequence encoding the extracellular domain of human IL-15Ra-E3, which lacks exon 3, (Anderson, D. et al., 1995, J. Biol. Chem.270:29862-29869) was fused to the 6× histidine tagged Fc of human IgG1via a polypeptide linker. The chimeric protein was expressed in Sf 21cells using a baculovirus expression system. Molecular Mass. Therecombinant mature human IL-15 Ra/Fc is a disulfide-linked homodimericprotein. Based on N-terminal sequencing, the recombinant human IL-15Raprotein has Ile 31 at the amino-terminus. The reduced human IL-15 Ra/Fcmonomer has a calculated molecular mass of approximately 42.6 kDa. As aresult of glycosylation, the recombinant monomer migrates as anapproximately 60-70 kDa protein in SDS-PAGE under reducing conditions.

Mouse. A DNA sequence encoding the signal peptide from human CD33,joined with amino acid residues 33-205 of the extracellular domain ofmouse IL-15 Ra (Giri, J. G. et al., 1995, EMBO. 14:3654-3663) was fusedto the Fc region of human IgG1 via a polypeptide linker. The chimericprotein was expressed in a mouse myeloma cell line, NS0. Molecular Mass.The recombinant mature mouse IL-15 Ra-Fc is a disulfide-linkedhomodimeric protein. Based on N-terminal sequencing, the recombinantmouse IL-15 Ra-Fc protein has Gly 33 at the amino-terminus. The reducedmouse IL-15 Ra-Fc monomer has a calculated molecular mass of 44.9 kDa.As a result of glycosylation, the recombinant protein migrates as anapproximately 80-90 kDa protein in SDS-PAGE under reducing conditions.In addition to the full-length IL-15 Ra-Fc, this preparation alsocontains a small amount (10%) of free IL-15 Ra and Fc generated byproteolytic cleavage. Free IL-15 Ra and Fc migrate as approximately 42kDa and 35 kDa proteins, respectively, in SDS-PAGE under reducingconditions.

CFSE labeling of cells and adoptive transfer. Lymphocytes were isolatedfrom spleen and/or peripheral lymph nodes (as described in Isolation oflymphocyte populations) and resuspended in HBSS (about 1% HGPG) at10×10⁶ cells/ml and then warmed to 37° C. Cells were incubated for about10 min with CFSE (0.01 mM; Molecular Probes, Eugene, Oreg.) and thereaction was squelched with HBSS with about 1% HGPG and about 5% FCS.Cells were washed twice with HBSS (about 1% HGPG). CFSE-labeled cellswere resuspended (1-20×10⁶ cells) in PBS and injected i.v. into congenicmice. Cells were isolated at the indicated times and analyzed for thepresence of donor cells using CD45 allele status and their expression ofsurface markers and CFSE intensity.

Isolation of lymphocyte populations and immunofluorescence analysis.Single-cell suspensions were created in HBSS (with about 1% HGPG) byhomogenizing spleens using frosted glass slides. Red blood cells werelysed and splenocytes were filtered through Nitex. At the indicated timepoints, lymphocytes were isolated and donor CFSE-labeled cells weredetected using their CD45 allele status or OVA-specific donor cells weredetected using an H-2 Kb tetramer containing the OVA-derived peptideSIINFEKL produced as previously described. For staining, lymphocyteswere suspended in PBS/about 0.2% BSA/about 0.1% NaN3 (FACS buffer) at aconcentration of about 3-15×10⁶/200 μl. When staining for tetramer,cells were incubated at room temperature for about 1 h with OVA-tetramerAPC plus the appropriate dilution of anti-CD8 PerCp. Cells were washedwith FACS buffer and stained with anti-CD44 PE at about 4° C. for about20 min, washed and then fixed in PBS with about 3% paraformaldehyde.Relative fluorescence intensities were measured with a FACScalibur (BDBiosciences, San Jose, Calif.). Data were analyzed using FlowJo Software(Tree Star, San Carlos, Calif.).

In vivo cytotoxicity assay. This assay was performed essentially aspreviously described. Normal spleen cells were labeled to low (about0.25 um) or high (about 2.5 um) CFSE levels and CFSEhigh cells wereincubated with about 1 μg/ml SIINFEKL peptide for about 45 min at about37° C. Equal numbers (10×10⁶) of each population were mixed and injectedi.v. into OT-I transferred mice that were either untreated or that weretreated with IL-15/IL-15Ra or were infected with 1×10⁵ pfu of vesicularstomatitis virus expressing chicken ovalbumin four days earlier. Fourhours later, spleen cells were analyzed for the presence of CFSEhigh andCFSElow populations Percent lysis=[1−(ratio unprimed/ratio primed)]×100.Ratio=percent CFSElow/percent CFSEhigh.

Intracellular detection of IFN-g. Lymphocytes were isolated from thespleen and cultured for about 5 h with about 1 μg/ml Golgistop (BDPharMingen), with or with about 1 μg/ml of the OVA-derived peptideSIINFEKL. After culture, cells were stained for surface molecules, thenfixed, and cell membranes were permeabilized in cytofix/cytopermsolution (BD PharMingen) and stained with anti-IFN-gPE or control ratIgG1 PE. Cells were then washed and the fluorescence intensity wasmeasured on a FACScalibur.

Bone marrow chimeras. Femurs and tibias were taken from CD11c-DTR Tg+mice or non-Tg littermates. The bone marrow (BM) was flushed out with asyringe and passed through a 70 um nylon mesh to generate a single cellsuspension. Red blood cells (RBC) were lysed and the cells resuspendedin HBSS supplemented with HEPES, L-glutamine, penicillin, streptomycin,gentamycin sulphate (HBSS-HGPG). To remove mature T cells from the BM,cells were incubated with anti-Thyl ascites fluid (T24), washed once inHBSS-HGPG then incubated with Low-Tox-M rabbit complement (CedarlaneLaboratories, Ontario, Canada) for about 45 min, at about 37° C. CD45.1recipient B6 mice were irradiated (about 1,000 rad) before about 2-5×10⁶bone marrow cells were transferred i.v. The mice were allowed to rest 8weeks before use. Diphtheria toxin (Sigma, St Louis, Mo.) in PBS wasadministered i.p. to mice at about 4 ng/g bodyweight. Chimeras receivedDT one day prior to cytokine treatment, a dose just prior to cytokinetreatment and a final dose on day three post cytokine treatment.

1.-19. (canceled)
 20. A method for modulating the immune response in anorganism comprising administration of an effective amount of apre-coupled polypeptide complex comprising an interleukin polypeptideportion with at least 90% homology to SEQ ID NO: 6, and an interleukinreceptor polypeptide portion with at least 90% homology to SEQ ID NO: 8,wherein the interleukin receptor polypeptide portion derived from SEQ IDNO: 8 is capable of binding said interleukin polypeptide portion derivedfrom SEQ ID NO:6.
 21. The method of claim 20, wherein the interleukin 15receptor alpha polypeptide is covalently linked to an antibody Fcportion.
 22. The method of claim 20, wherein aid interleukin 15polypeptide portion further comprises a heterologous signal peptidesequence on the amino terminus.
 23. The method of claim 20, wherein thecomplex comprises the interleukin 15 polypeptide and the interleukin 15receptor alpha polypeptide within a single polypeptide chain.
 24. Themethod of claim 20, wherein the IL-15 polypeptide does not contain asignal sequence.
 25. The method of claim 20, wherein the polypeptide thepre-coupled complex has an in vivo half-life of at least one-hour. 26.The method of claim 20, wherein the polypeptide complex increases T cellproliferation.
 27. The method of claim 20, wherein the polypeptidecomplex enhances immune function in said organism in response tocancers.
 28. The method of claim 20, wherein the polypeptide complexenhances immune function in said organism in response to viral orbacterial infections.
 29. The method of claim 20, wherein thepolypeptide complex is administered in combination with a therapeuticagent, wherein the therapeutic agent is an anti-oncogenic agent.
 30. Themethod of claim 20, wherein the polypeptide complex is administered incombination with at least one pharmaceutically acceptable excipient,adjuvant, biologically active agent.